High Definition Microdroplet Printer

ABSTRACT

Methods for delivering discrete entities including, e.g., cells, media or reagents to substrates are provided. In certain aspects, the methods include manipulating and/or analyzing qualities of the entities or biological components thereof. In some embodiments, the methods may be used to create arrays of microenvironments and/or for two and three-dimensional printing of tissues or structures. Systems and devices for practicing the subject methods are also provided.

CROSS-REFERENCE

This application claims priority benefit of U.S. Provisional ApplicationNo. 62/112,068, filed Feb. 4, 2015, and U.S. Provisional Application No.62/067,314, filed Oct. 22, 2014, which applications are incorporatedherein by reference in their entireties and for all purposes.

GOVERNMENT SUPPORT

This invention was made with government support under grant numbers1R21HG007233-02, 1DP2AR068129-01, and 1R01EB019453-01 awarded by theNational Institutes of Health, grant numbers HR0011-12-C-0065,N66001-12-C-4211, and HR0011-12-C-0066 awarded by the Department ofDefense, and grant number DBI-1253293 awarded by the National ScienceFoundation. The government has certain rights in the invention.

INTRODUCTION

Developments in droplet microfluidics have provided a robust tool setfor the high-throughput manipulation and analysis of single cells andsmall reagent volumes. However, measurements and droplet manipulationsare generally performed on droplets flowing single file throughsub-regions of a microfluidic device, thus providing a limited abilityto perform measurements over extended periods of time or to maketargeted reagent additions to specific droplets.

SUMMARY

Methods for delivering discrete entities including, e.g., cells, mediaand/or reagents encapsulated therein to substrates are provided. Incertain aspects, the methods include manipulating and/or analyzingqualities of the discrete entities or biological materials encapsulatedtherein. In some embodiments, the methods may be used to create arraysof microenvironments and/or for two and three-dimensional printing oftissues or structures. Systems and devices for practicing the subjectmethods are also provided.

The present disclosure provides methods of delivering discrete entitiesto a substrate, for example, by: flowing a plurality of discreteentities through a microfluidic device in a carrier fluid, wherein thediscrete entities are insoluble and/or immiscible in the carrier fluid;directing the carrier fluid and one or more of the plurality of discreteentities through a delivery orifice to the substrate; and affixing theone or more of the plurality of discrete entities to the substrate.

The present disclosure also provides methods of printing one or morecell layers, for example, by: encapsulating cells in droplets includingan aqueous fluid to provide cell-comprising droplets; flowing aplurality of droplets comprising the cell-comprising droplets through amicrofluidic device in a carrier fluid, wherein the carrier fluid isimmiscible with the aqueous fluid; directing the carrier fluid and aplurality of the cell-comprising droplets through a delivery orifice toa substrate; and affixing the plurality of the cell-comprising dropletsto the substrate to provide a first layer of cell-comprising droplets,wherein the substrate comprises on a first surface a layer of fluidwhich is miscible with the carrier fluid and immiscible with the aqueousfluid, and wherein the plurality of the cell-comprising droplets areaffixed to the first surface of the substrate following introductioninto the layer of fluid on the first surface of the substrate.

The present disclosure also provides methods of printing and detectingone or more cells, for example, by: encapsulating cells in dropletsincluding an aqueous fluid to provide cell-comprising droplets; flowinga plurality of droplets comprising the cell-comprising droplets througha microfluidic device in a carrier fluid, wherein the carrier fluid isimmiscible with the aqueous fluid; directing the carrier fluid and aplurality of the cell-comprising droplets through a delivery orifice tothe substrate; affixing the plurality of the cell-comprising droplets tothe substrate, wherein the substrate includes on a first surface a layerof fluid which is miscible with the carrier fluid and immiscible withthe aqueous fluid, and wherein the plurality of the cell-comprisingdroplets are affixed to the first surface of the substrate followingintroduction into the layer of fluid on the first surface of thesubstrate; and detecting one or more of the cells in the affixedcell-comprising droplets, a component of one or more of the cells in theaffixed cell-comprising droplets, or a product of one or more of thecells in the affixed cell-comprising droplets.

The present disclosure also provides methods of printing athree-dimensional structure, for example, by: flowing discrete entitiesthrough a microfluidic device in a carrier fluid, wherein the discreteentities are insoluble and/or immiscible in the carrier fluid; anddirecting the carrier fluid and a first plurality of the discreteentities through a delivery orifice to a substrate to provide a firstlayer thereon; directing the carrier fluid and a second plurality of thediscrete entities through the delivery orifice to the first layer toprovide a second layer thereon; and one or more additional directingsteps in which a plurality of the discrete entities are directed throughthe delivery orifice to an immediately preceding layer to provide asubsequent layer thereon, wherein a multilayer, three-dimensionalstructure is provided.

The present disclosure also provides methods of delivering droplets froma delivery orifice, for example, by: flowing a plurality of dropletsthrough a microfluidic device in a carrier fluid, wherein themicrofluidic device includes a sorter; detecting one or more of theplurality of droplets to provide one or more detected droplets; sortingvia the sorter the one or more detected droplets from the plurality ofdroplets; and directing the carrier fluid and the one or more detecteddroplets through the delivery orifice.

The present disclosure also provides methods of affixing a droplet to asubstrate, for example, by: delivering a droplet in a first carrierfluid from a microfluidic device, through an orifice, to a substratesurface; positioning the droplet in a second carrier fluid on thesubstrate surface; and affixing the droplet to the substrate surface viaa force.

The present disclosure also provides methods of moving an affixeddroplet on a substrate, for example, by: delivering a droplet in a firstcarrier fluid from a microfluidic device, through an orifice, to asubstrate surface; positioning the droplet in a second carrier fluid onthe substrate surface; affixing the droplet to the substrate surface viaa force; and modulating the force so as to move the droplet from itsaffixed location to another location and/or applying a second force,which is sufficient, either alone or in combination with the modulatedforce, to move the droplet from its affixed location to anotherlocation.

The present disclosure also provides methods of adding reagents to adroplet, for example, by: delivering a first droplet in a first carrierfluid from a microfluidic device, through an orifice, to a substratesurface; positioning the droplet in a second carrier fluid on thesubstrate surface; affixing the droplet to the substrate surface via aforce; delivering a second droplet to the same location as the firstdroplet affixed to the substrate surface or a location adjacent orproximate the first droplet on the substrate surface; and coalescing thefirst droplet and the second droplet such that the contents of the firstdroplet and the second droplet are combined.

The present disclosure also provides methods of adding reagents to adroplet, for example, by: delivering a droplet in a first carrier fluidfrom a microfluidic device, through a first orifice, to a substratesurface; positioning the droplet in a second carrier fluid on thesubstrate surface; affixing the droplet to the substrate surface via aforce; inserting a second orifice fluidically connected to a reagentsource into the droplet; and injecting via the second orifice one ormore reagents into the droplet.

The present disclosure also provides methods of recovering all or aportion of an affixed droplet, for example, by: delivering a droplet ina first carrier fluid from a microfluidic device, through an orifice, toa substrate surface; positioning the droplet in a second carrier fluidon the substrate surface; affixing the droplet to the substrate surfacevia a force; and recovering all or a portion of the affixed droplet.

The present disclosure also provides methods of manipulating an affixeddroplet, for example, by: delivering a droplet in a first carrier fluidfrom a microfluidic device, through an orifice, to a substrate surface;positioning the droplet in a second carrier fluid on the substratesurface; affixing the droplet to the substrate surface via a force; andmodulating the immediate environment of the droplet, thereby modulatingthe contents of the droplet.

The present disclosure also provides methods of manipulating an affixeddroplet by delivering a droplet in a first carrier fluid from amicrofluidic device, through an orifice, to a substrate surface;positioning the droplet in a second carrier fluid on the substratesurface; affixing the droplet to the substrate surface via a force; atleast partially solidifying the affixed droplet; removing the secondcarrier fluid from the substrate surface, wherein the second carrierfluid is immiscible with the contents of the affixed droplet prior tothe at least partial solidification of the affixed droplet; replacingthe removed second carrier fluid with a miscible fluid; and modulating achemical composition of the miscible fluid, thereby modulating theaffixed droplet.

The present disclosure also provides methods of porating a cell withinan affixed droplet, for example, by: delivering a droplet in a firstcarrier fluid from a microfluidic device, through an orifice, to asubstrate surface, wherein the droplet includes a cell; positioning thedroplet in a second carrier fluid on the substrate surface; affixing thedroplet to the substrate surface via a force; and porating the cellwithin the droplet.

The present disclosure also provides methods of analyzing a droplet on asubstrate, for example, by: delivering a droplet in a first carrierfluid from a microfluidic device, through an orifice, to a substratesurface; positioning the droplet in a second carrier fluid on thesubstrate surface; affixing the droplet to the substrate surface via aforce; and detecting one or more components of the affixed droplet.

The present disclosure also provides methods of delivering discreteentities to a substrate, for example, by: flowing a plurality of firstdiscrete entities through a first microfluidic device in a first carrierfluid, wherein the first discrete entities are insoluble and/orimmiscible in the first carrier fluid, and wherein the firstmicrofluidic device includes a first delivery orifice; directing thefirst carrier fluid and one or more of the plurality of first discreteentities through the first delivery orifice to the substrate; flowing aplurality of second discrete entities through a second microfluidicdevice in a second carrier fluid, wherein the second discrete entitiesare insoluble and/or immiscible in the second carrier fluid, and whereinthe second microfluidic device includes a second delivery orifice;directing the second carrier fluid and one or more of the plurality ofsecond discrete entities through the second delivery orifice to thesubstrate; and affixing the one or more of the plurality of firstdiscrete entities and the one or more of the plurality of seconddiscrete entities to the substrate.

The present disclosure also provides methods of delivering discreteentities to a substrate, for example, by: flowing a plurality ofdiscrete entities through a microfluidic device in a carrier fluid,wherein the discrete entities are insoluble and/or immiscible in thecarrier fluid, and wherein the microfluidic device includes a pluralityof delivery orifices; directing the carrier fluid and a first one ormore of the plurality of discrete entities through a first deliveryorifice of the plurality of delivery orifices to the substrate;directing the carrier fluid and a second one or more of the plurality ofdiscrete entities through a second delivery orifice of the plurality ofdelivery orifices to the substrate; and affixing the first one or moreof the plurality of first discrete entities and the second one or moreof the plurality of discrete entities to the substrate.

The present disclosure also provides methods of analyzing a droplet, forexample, by: flowing a plurality of droplets through a microfluidicdevice in a carrier fluid, encapsulating or incorporating uniqueidentifier molecules into the plurality of droplets, such that eachdroplet of the plurality of droplets includes a different uniqueidentifier molecule; delivering the plurality of droplets in a firstcarrier fluid from a microfluidic device, through an orifice, to asubstrate surface; positioning the plurality of droplets in a secondcarrier fluid on the substrate surface; affixing the plurality ofdroplets to the substrate surface via a force; for each of the affixedplurality of droplets, recovering all or a portion of the affixeddroplet and the unique identifier for each droplet; analyzing therecovered droplets or recovered portions thereof in conjunction with theunique identifier, wherein results of the analysis are identified asspecific to material originating from particular droplets based on thepresence of the unique identifier.

The present disclosure also provides methods of performing quantitativePCR, for example, by: partitioning a heterogeneous population of nucleicacids into a plurality of droplets including an aqueous fluid;encapsulating or incorporating quantitative PCR reagents into theplurality of droplets; flowing the plurality of droplets through amicrofluidic device in a carrier fluid, wherein the carrier fluid isimmiscible with the aqueous fluid; directing the carrier fluid and aplurality of droplets through a delivery orifice to a substrate;affixing the plurality of droplets to the substrate, wherein thesubstrate includes on a first surface a layer of fluid which is misciblewith the carrier fluid and immiscible with the aqueous fluid, andwherein the plurality of droplets are affixed to the first surface ofthe substrate following introduction into the layer of fluid on thefirst surface of the substrate; incubating the affixed plurality ofdroplets under conditions sufficient for amplification of nucleic acids;and detecting nucleic acid amplification over time.

The present disclosure also provides methods of sequencing single cellnucleic acids, for example, by: partitioning a heterogeneous pluralityof cells into a plurality of droplets including an aqueous fluid, suchthat each droplet includes not more than one cell; subjecting theplurality of droplets to conditions sufficient for lysis of the cellscontained therein and release of cellular nucleic acids; encapsulatingor incorporating unique nucleic acid identifier molecules into theplurality of droplets, such that each droplet of the plurality ofdroplets includes a different unique nucleic acid identifier molecule;linking the unique nucleic acid identifier molecules to one or morecellular nucleic acids in the plurality of droplets or to amplificationproducts thereof; flowing the plurality of droplets through amicrofluidic device in a first carrier fluid; delivering the pluralityof droplets in the first carrier fluid from the microfluidic device,through an orifice, to a substrate surface; positioning the plurality ofdroplets in a second carrier fluid on the substrate surface; affixingthe plurality of droplets to the substrate surface via a force; for eachof the affixed plurality of droplets, recovering all or a portion of theaffixed droplet, including cellular nucleic acids and the unique nucleicacid identifier for each droplet; sequencing nucleic acids from therecovered droplets or recovered portions thereof together with theunique identifier molecules, wherein the presence of the sequence of aunique identifier molecule in the sequence read of a nucleic acidmolecule identifies the nucleic acid molecule as originating from aparticular cell.

The present disclosure also provides methods of synthesizing a polymeron a substrate, for example, by: flowing a first droplet including afirst droplet fluid through a microfluidic device in a carrier fluid,wherein the first droplet includes a first polymer or a first monomer;directing the carrier fluid and the first droplet through a deliveryorifice to the substrate; affixing the first droplet to the substratewherein the substrate includes on a first surface a layer of fluid whichis miscible with the carrier fluid and immiscible with the first dropletfluid, and wherein the first droplet is affixed to the first surface ofthe substrate at a predetermined location following introduction intothe layer of fluid on the first surface of the substrate; flowing asecond droplet through the microfluidic device in the carrier fluid,wherein the second droplet includes a second polymer or a secondmonomer; directing the carrier fluid and the second droplet through thedelivery orifice to the first droplet affixed at the predeterminedlocation; incubating the first and second droplets under conditionssufficient for the contents of the first and second droplets to comeinto contact and for the first polymer or first monomer to form acovalent bond with the second polymer or monomer, thereby generating asynthesized polymer.

The present disclosure also provides methods of analyzing a droplet on asubstrate, for example, by: partitioning a molecular library including aplurality of library members into a plurality of droplets including anaqueous fluid; delivering the plurality of droplets in a first carrierfluid from a microfluidic device, through an orifice, to a substratesurface; positioning the droplets in a second carrier fluid on thesubstrate surface; affixing the droplets to the substrate surface via aforce; and performing one or more reactions in the affixed droplets withthe library members; detecting the results of the one or more reactionsin the affixed droplets and/or recovering all or a portion of theaffixed droplets for further analysis.

The present disclosure also provides methods of printing microarrays,for example, by: delivering a plurality of droplets in a first carrierfluid from a microfluidic device, through an orifice, to a substratesurface, wherein each of the plurality of droplets includes a molecule;positioning the droplets in a second carrier fluid on the substratesurface; affixing the droplets at predetermined locations to thesubstrate surface via a force; incubating the substrate under conditionssuitable for chemical bonding of the molecules comprised by the affixeddroplets to the substrate surface, thereby providing an array ofsubstrate-bound molecules.

The present disclosure also provides methods of performing in situsequencing, for example, by: flowing a plurality of droplets through amicrofluidic device in a carrier fluid, encapsulating or incorporatingunique nucleic acid identifier molecules into the plurality of droplets,such that each droplet of the plurality of droplets includes one or morecopies of a different unique nucleic acid identifier molecule;delivering the plurality of droplets in a first carrier fluid from amicrofluidic device, through an orifice, to a surface of a tissuesubstrate; positioning the plurality of droplets in a second carrierfluid on the surface of the tissue substrate; affixing the plurality ofdroplets to the surface of the tissue substrate via a force; incubatingthe tissue substrate under conditions sufficient for the unique nucleicacid identifier molecules from each affixed droplet to bind to nucleicacids contained within the tissue substrate in proximity to the affixeddroplet; sequencing the unique nucleic acid identifier molecules and thenucleic acids to which they are bound; and identifying and/orquantitating, using the unique nucleic acid identifier molecules,nucleic acids contained within the tissue substrate at locationscorresponding to locations where particular droplets were affixed.

The present disclosure also provides methods of manipulating cells orembryos, for example, by: flowing a plurality of droplets through amicrofluidic device in a carrier fluid, wherein each droplet of theplurality of droplets includes an aqueous fluid and a fertilized eggcell or embryo, and wherein the carrier fluid is immiscible with theaqueous fluid; directing the carrier fluid and the plurality of dropletsthrough a delivery orifice to a substrate; affixing the plurality ofdroplets to the substrate, wherein the substrate includes on a surfacethereof a layer of fluid which is miscible with the carrier fluid andimmiscible with the aqueous fluid, and wherein the plurality of dropletsis affixed to the surface of the substrate following introduction intothe layer of fluid on the surface of the substrate; detecting within theaffixed plurality of droplets the development of one or more embryos;and selecting and recovering an embryo from the affixed droplets.

The present disclosure also provides methods of manipulating cells orembryos, for example, by: flowing a plurality of droplets through amicrofluidic device in a carrier fluid, wherein each droplet of theplurality of droplets includes an aqueous fluid and an unfertilized eggcell, and wherein the carrier fluid is immiscible with the aqueousfluid; directing the carrier fluid and the plurality of droplets througha delivery orifice to a substrate; fertilizing one or more of the eggcells in the plurality of droplets; affixing the plurality of dropletsto the substrate, wherein the substrate includes on a surface thereof alayer of fluid which is miscible with the carrier fluid and immisciblewith the aqueous fluid, and wherein the plurality of droplets areaffixed to the surface of the substrate following introduction into thelayer of fluid on the surface of the substrate; detecting within theaffixed droplets the development of an embryo; and selecting andrecovering specific embryos from the affixed droplets.

The present disclosure also provides systems and devices which may beutilized in the implementation of the methods describe herein. Forexample, the present disclosure provides a droplet printer including,for example: a microfluidic device including one or more droplet makersand one or more flow channels, wherein the one or more flow channels arefluidically connected to the one or more droplet makers and configuredto receive one or more droplets therefrom; a delivery orificefluidically connected to one or more of the one or more flow channels;and an automated system integrated with the delivery orifice, whereinthe automated system (a) selectively positions the delivery orifice inproximity to a substrate during operation or (b) selectively positionsthe substrate in proximity to the delivery orifice during operation,such that a droplet can be ejected from the delivery orifice anddeposited on the substrate.

The present disclosure also provides a system including, for example, adroplet printer including a substrate surface for receiving one or moredroplets deposited by the delivery orifice of the droplet printer; andone or more of: (a) a temperature control module operably connected tothe droplet printer, (b) a detection means operably connected to thedroplet printer, (c) an incubator operably connected to the dropletprinter, and (d) a sequencer operably connected to the droplet printer;and a conveyor configured to convey the substrate from a first dropletreceiving position to one or more of (a)-(d).

The present disclosure also provides substrates which may be providedusing the methods, devices and systems described herein, for example, asubstrate including: a substrate surface comprising an immiscible phasefluid; and an ordered array of droplets positioned in the immisciblephase fluid, wherein the droplets are affixed to the substrate surface,and wherein the ordered array of droplets comprises at least 10,000individual droplets.

The present disclosure also provides electrode array systems, forexample, an electrode array system including: an array of individuallycontrollable electrodes embedded in a substrate material; a powersource; and a controller, wherein the controller is configured toselectively enable or disable an electrical connection between the powersource and each individually controllable electrode in the array therebyproviding an active an inactive electrode respectively, and wherein,each active electrode is capable of affixing a discrete entity to asurface of the substrate material in proximity to the active electrodewhen said discrete entity is deposited in proximity to the activeelectrode.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention may be best understood from the following detaileddescription when read in conjunction with the accompanying drawings.Included in the drawings are the following figures:

FIG. 1 provides a simplified depiction of a microfluidic system andmethod of the instant disclosure.

FIG. 2 depicts one embodiment of a subject device and associatedmethods, including methods of sorting discrete entities. An embodimentof a reinjection junction, sorting junction and a process of sorting bymaking a positive or negative sort are specifically illustrated inpanels 1-3.

FIG. 3 provides a simplified representation of one type of microfluidicsystem and an associated method of the present disclosure. Theapplication depicted is the delivery of discrete entities includingcells to a substrate.

FIG. 4 illustrates aspects of the subject devices and methods includinga substrate designed with electrode geometry configured to impart highelectric field gradients on the surface of the substrate, creatingdielectrophoretic forces that pull droplets towards the substratesurface. An exemplary device depositing aqueous droplets on thesubstrate surface is shown. After contact with the surface, theapplication of electric fields destabilizes the thin film of immisciblecarrier fluid, e.g., oil, separating the aqueous droplets from thesubstrate surface, causing the droplets to wet the surface. Once wetted,droplets further flatten in a manner that correlates to the applieddielectrophoretic force. High electric field gradients are created bygeometries where charged features are separated by small distances. Thiscan be created by exciting one feature with an AC signal and groundingthe counter-feature. If the counter-feature is not grounded, highelectric field gradients may still be generated because electromagneticscreening (charge reorganization) in the counter-feature causes it toact similarly to a grounded channel.

FIG. 5 illustrates aspects of the subject devices and methods includinga patterned substrate having unfilled positions where discrete entitiesmay be affixed and filled positions where discrete entities are affixed.

FIG. 6, Panels A-C, illustrate one embodiment of affixing a discreteentity, e.g., a droplet, generated in a microfluidic device to asubstrate by applying a force, e.g., a dielectrophoretic force. Panel Ashows a droplet being ejected from a microfluidic nozzle. Panel B showsthe droplet caught at a gap separating electrode features. Panel Cillustrates stretching of the trapped droplet via the application ofdielectrophoretic force.

FIG. 7, Panels A-D, illustrate aspects of well plates and projectedarray densities which may be achieved using the methods, devices andsystems of the present disclosure. Panel A: A standard 384 well plate.Panel B: A “well” plate which may be generated using the subjectmethods. Panel C: Side and top views of a substrate having of fourproximately-affixed droplets thereon. Panel D: A graph demonstrating howthe subject methods, for example using picodrop printing, can increasethe array density within a standard well plate footprint.

FIG. 8 provides a schematic of a system according to the presentdisclosure. A droplet microfluidic print head, including a compactmicrofluidics droplet sorter modified with an exit nozzle, is suspendedabove the stage of an inverted microscope. Droplets flowing through thesorter are fluorescently labeled and detected within the device by alaser coupled to external detection optics. When a desired droplet isdetected, it is actively sorted to the nozzle and directed to a targetsurface. A constant background flow of carrier fluid (e.g., oil) bringsthe droplet in close contact with the dielectrophoretic trap. Acustomized substrate with biopolar electrodes patterned into its surfaceis placed on the xy stage of the microscope and serves as a target forthe deposition of droplets. Specific regions on the substrate with highelectric field gradients serve as dieletrophoretic traps for droplets bycausing movement of droplets towards, and wetting onto these regions. Inthis implementation, the nozzle of the print head is held stationary,while the substrate is translated horizontally.

FIG. 9 provides an image from a droplet printing example. The print headand the network of dielectrophoretic traps are visible and in the planeof the image. A series of droplets printed to the surface are visiblealong the top of the image.

FIG. 10, Panels A-C, provide (a) a schematic of a droplet sorteraccording to an embodiment of the present disclosure with detected andselectively displaced black droplets being separated by a gapped dividerof reduced channel height. (b) Still from high speed video of 22 kHzsorting. With a conventional hard wall divider, droplets not fullydisplaced are split (c), while larger applied dielectrophoretic forcespull droplets apart (d). Scale bars are 50 μm.

FIG. 11, Panels A-E, provide (a) a schematic of a microfluidic deviceaccording to an embodiment of the present disclosure, with shallowchannels in boxed regions indicating areas magnified in other figures.Microscope images of (b) irregularly spaced, reinjected droplets from asingle-layer reinjector and (c) regularly spaced droplets from theactual two-layer reinjector used to sort. (d) The droplet filter beforethe reinjector. (e) Equilibration channels connecting the exit outlets.Scale bars are 500 μm in (a) and 50 μm in (b-e).

FIG. 12, Panels A-D, provide (a) Time series during a sort showing thePMT-detected fluorescence signal (blue) as well as the voltage appliedto the electrode (red). Inset shows the frequency components from aFourier transform of a longer time series during the same sort, as wellas the range of previously reported sort rates. Fluorescence microscopeimages, also from the same sort, of the pre-sorted droplets (6.4%bright), the positive droplets (99.3% bright), and negative droplets(0.2% bright). Scale bars are 100 μm.

DETAILED DESCRIPTION

Methods for delivering discrete entities including, e.g., cells, mediaand/or reagents encapsulated therein to substrates are provided. Incertain aspects, the methods include manipulating and/or analyzingqualities of the discrete entities or biological materials encapsulatedtherein. In some embodiments, the methods may be used to create arraysof microenvironments and/or for two and three-dimensional printing oftissues or structures. Systems and devices for practicing the subjectmethods are also provided.

The subject methods and devices may find use in a wide variety ofapplications, such as increasing the accuracy and/or efficiency ofprinting, e.g., microdroplet printing, and in assays involving, forexample, well-plate analysis. Assays which can be performed inaccordance with the subject disclosure may be relevant for the detectionof cancer or other diseases, monitoring disease progression, analyzingthe DNA or RNA content of cells, and a variety of other applications inwhich it is desired to detect and/or quantify specific components of adiscrete entity.

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andexemplary methods and materials may now be described. Any and allpublications mentioned herein are incorporated herein by reference todisclose and describe the methods and/or materials in connection withwhich the publications are cited. It is understood that the presentdisclosure supersedes any disclosure of an incorporated publication tothe extent there is a contradiction.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “adroplet” includes a plurality of such droplets and reference to “thediscrete entity” includes reference to one or more discrete entities,and so forth.

It is further noted that the claims may be drafted to exclude anyelement, e.g., any optional element. As such, this statement is intendedto serve as antecedent basis for use of such exclusive terminology as“solely”, “only” and the like in connection with the recitation of claimelements, or the use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Further,the dates of publication provided may be different from the actualpublication dates which may need to be independently confirmed. To theextent the definition or usage of any term herein conflicts with adefinition or usage of a term in an application or referenceincorporated by reference herein, the instant application shall control.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

Methods

As summarized above, aspects of the disclosed subject matter includemethods for the delivery of discrete entities, such as droplets, to oneor more substrates and in some embodiments, affixing the discreteentities thereto. Aspects of the present disclosure include methods forprinting one or more medium or cell layers as well as the detection ofone or more qualities of components which are applied to a substrate.For example, some embodiments include methods for the detection,quantification, and/or genotyping of cells, e.g. normal cells (i.e.,non-tumor cells), or tumor cells positioned on a substrate.

The subject methods, in some embodiments, include flowing one or morediscrete entities through a microfluidic device in a carrier fluid, suchas a carrier fluid in which the discrete entities are insoluble and/orimmiscible. The methods also may include directing the carrier fluid andone or more of the discrete entities through a portion of a microfluidicdevice, such as a delivery orifice, to a substrate and/or affixing theone or more discrete entities to a substrate. Discrete entities may beaffixed to a substrate, for example, by one or more forces, such as anelectrical (e.g., dielectrophoretic), gravitational, and/or magneticforce.

Discrete entities as used or generated in connection with the subjectmethods, devices, and/or systems may be sphere shaped or they may haveany other suitable shape, e.g., an ovular or oblong shape. Discreteentities as described herein may include a liquid phase and/or a solidphase material. In some embodiments, discrete entities according to thepresent disclosure include a gel material. In some embodiments, thesubject discrete entities have a dimension, e.g., a diameter, of orabout 1.0 μm to 1000 μm, inclusive, such as 1.0 μm to 750 μm, 1.0 μm to500 μm, 1.0 μm to 100 μm, 1.0 μm to 10 μm, or 1.0 μm to 5 μm, inclusive.In some embodiments, discrete entities as described herein have adimension, e.g., diameter, of or about 1.0 μm to 5 μm, 5 μm to 10 μm, 10μm to 100 μm, 100 μm to 500 μm, 500 μm to 750 μm, or 750 μm to 1000 μm,inclusive. Furthermore, in some embodiments, discrete entities asdescribed herein have a volume ranging from about 1 fL to 1 nL,inclusive, such as from 1 fL to 100 pL, 1 fL to 10 pL, 1 fL to 1 pL, 1fL to 100 fL, or 1 fL to 10 fL, inclusive. In some embodiments, discreteentities as described herein have a volume of 1 fL to 10 fL, 10 fL to100 fL, 100 fL to 1 pL, 1 pL to 10 pL, 10 pL to 100 pL or 100 pL to 1nL, inclusive. In addition, discrete entities as described herein mayhave a size and/or shape such that they may be produced in, on, or by amicrofluidic device and/or flowed from or applied by a microfluidicdevice.

In some embodiments, the discrete entities as described herein aredroplets. The terms “drop,” “droplet,” and “microdroplet” are usedinterchangeably herein, to refer to small, generally sphericallystructures, containing at least a first fluid phase, e.g., an aqueousphase (e.g., water), bounded by a second fluid phase (e.g., oil) whichis immiscible with the first fluid phase. In some embodiments, dropletsaccording to the present disclosure may contain a first fluid phase,e,g, oil, bounded by a second immiscible fluid phase, e.g. an aqueousphase fluid (e.g, water). In some embodiments, the second fluid phasewill be an immiscible phase carrier fluid. Thus droplets according tothe present disclosure may be provided as aqueous-in-oil emulsions oroil-in-aqueous emulsions. Droplets may be sized and/or shaped asdescribed herein for discrete entities. For example, droplets accordingto the present disclosure generally range from 1 μm to 1000 μm,inclusive, in diameter. Droplets according to the present disclosure maybe used to encapsulate cells, nucleic acids (e.g., DNA), enzymes,reagents, and a variety of other components. The term droplet may beused to refer to a droplet produced in, on, or by a microfluidic deviceand/or flowed from or applied by a microfluidic device.

As used herein, the term “carrier fluid” refers to a fluid configured orselected to contain one or more discrete entities, e.g., droplets, asdescribed herein. A carrier fluid may include one or more substances andmay have one or more properties, e.g., viscosity, which allow it to beflowed through a microfluidic device or a portion thereof, such as adelivery orifice. In some embodiments, carrier fluids include, forexample: oil or water, and may be in a liquid or gas phase. Suitablecarrier fluids are described in greater detail herein.

FIG. 1 presents a non-limiting, simplified representation of one type ofa microfluidics system and method according to the present disclosure.The particular embodiment depicted in FIG. 1 shows the delivery ofdiscrete entities (droplets are illustrated by way of example) to asubstrate. In one such method, discrete entities 101, e.g., droplets,are prepared using a device, e.g., a microfluidic device 100, and acarrier fluid 102 to produce a mixed emulsion including the discreteentities. A variety of suitable droplet makers are known in the art,which may be used to prepare the mixed emulsion, e.g., droplet makersdescribed in PCT Publication No. WO 2014/028378, the disclosure of whichis incorporated by reference herein in its entirety and for allpurposes. In some embodiments, the discrete entities are of more thanone type, e.g., more than one composition and/or size, such as a firsttype, e.g., a type containing one or more cells of interest, and asecond type, e.g., a type not containing one or more cells of interest.In some embodiments, the discrete entities may contain one or morebeads, such as magnetic beads and/or conductive beads.

In some embodiments of the disclosed methods, microfluidic devices areutilized which include one or more droplet makers configured to formdroplets from a fluid stream. Suitable droplet makers includeselectively activatable droplet makers and the methods may includeforming one or more discrete entities via selective activation of thedroplet maker. The methods may also include forming discrete entitiesusing a droplet maker, wherein the discrete entities include one or moreentities which differ in composition.

Once prepared, a mixed emulsion may be moved, e.g., moved and/or flowedto another portion of the microfluidic device 100, such as a sorter 103.A subset of the discrete entities 101 may be separated using a sorter103. A sorter 103 may be configured to detect and/or separate discreteentities, e.g., discrete entities present in a carrier fluid, havingdifferent types, e.g., different compositions and/or sizes, such as afirst type, e.g., a type containing one or more cells of interest, and asecond type, e.g., a type not containing one or more cells of interest.As such, a sorter 103 may provide one or more sorted discrete entities106 (e.g., one or more discrete entities including a cell and/or nucleicacid of interest) and direct them via a first channel 104 to a nozzleincluding a delivery orifice 107 for delivery to a substrate 108. Asorter 103 may also provide one or more sorted discrete entities 112(e.g., one or more discrete entities not including a cell and/or nucleicacid of interest) and direct them via a second channel 105 to a wasteoutlet.

In some embodiments, the discrete entities not sorted for delivery via adelivery orifice, are recovered and/or recycled by, for example, beingre-injected into the carrier fluid upstream of the sorter 103. Variousembodiments of the methods disclosed herein include repeated recyclingof discrete entities not selected for delivery through the deliveryorifice in a particular pass through the sorter. Sorting, according tothe subject embodiments, is described in further detail below. Also, invarious embodiments, one or more discrete entities, e.g., all thediscrete entities present in a mixed emulsion, remain contained e.g.,encapsulated, in a carrier fluid, e.g., a hydrophobic solution (e.g.,oil), or a hydrophilic solution (e.g., an aqueous solution), prior tosorting and/or throughout a sorting process carried out by the sorter103 and/or throughout the process of directing the one or more entitiesthrough a portion of a microfluidic device, e.g., a delivery orifice,and/or throughout a process of affixing the entities to a substrate.

As discussed above, a sorted subset of discrete entities of interest,e.g., discrete entities 106, (e.g., discrete entities containing one ormore cells of interest), may in some embodiments, be directed through adelivery orifice 107 of a microfluidic device 100 to a substrate 108. Insome embodiments of the methods, a microfluidic device 100, or a portionthereof, e.g., a delivery orifice 107, contacts a substrate, e.g., asubstrate 108, or a portion thereof, to which it delivers discreteentities. In other embodiments, a microfluidic device 100, or a portionthereof, e.g., a delivery orifice 107, delivers discrete entities to asubstrate, e.g., a substrate 108, or a portion thereof, by dispensingthe discrete entities in a carrier fluid, e.g., a carrier fluid 102, inproximity to a surface of the substrate, for example into a fluid on thesurface of the substrate (e.g., substrate fluid 110), which fluid ismiscible with the carrier fluid and immiscible with the discreteentities.

A delivery orifice as described herein, e.g., a delivery orifice of amicrofluidic nozzle as described herein, will generally have dimensionsthat are similar to the size of the droplets to be deliveredtherethrough. Accordingly, in some embodiments, a delivery orifice asdescribed herein has a diameter of from about 1 μm to about 1000 μm,inclusive, e.g., from about 10 μm to about 300 μm, inclusive. In someembodiments, a delivery orifice as described herein has a diameter offrom about 1 μm to about 10 μm, from about 10 μm to about 100 μm, fromabout 100 μm to about 500 μm, or from about 500 μm to about 1000 μm,inclusive.

The nozzle can be molded as part of a microfluidic sorter as describedherein, or can be a separate part that is mated with a microfluidicsorter as described herein. Suitable materials for the nozzle mayinclude, e.g., polymeric tubing, small bore hypodermic tubing, andmodified glass capillaries.

One embodiment of the subject systems, devices and methods is nowdescribed with reference to FIG. 2, which illustrates a microfluidicsystem including a microfluidic device including a sorting junction. Asshown in FIG. 2, a microfluidic device is employed to apply and sort amixed emulsion in order to deliver select discrete entities, e.g.,droplets, to a delivery orifice, e.g., a delivery orifice of a printhead. The microfluidic device utilizes a moat salt solution (to generatethe field gradient used for dielectrophoretic deflection and to limitstray fields that can cause unintended droplet merger), spacer oil, andan electrode salt solution to facilitate sorting. The microfluidicdevice depicted provides junctions including a reinjection junction forproviding a discrete entity-containing emulsion to be sorted and asorting junction including a sorter for sorting, e.g., by makingpositive and negative sorts of discrete entities. Sorting may beaccomplished, e.g., by applying an electric field via an electrode,e.g., a liquid electrode including, e.g., an electrode salt solution.

In certain embodiments, liquid electrodes include liquid electrodechannels filled with a conducting liquid (e.g. salt water or buffer) andsituated at positions in the microfluidic device where an electric fieldis desired. In particular embodiments, the liquid electrodes areenergized using a power supply or high voltage amplifier. In someembodiments, the liquid electrode channel includes an inlet port so thata conducting liquid can be added to the liquid electrode channel. Suchconducting liquid may be added to the liquid electrode channel, forexample, by connecting a tube filled with the liquid to the inlet portand applying pressure. In particular embodiments, the liquid electrodechannel also includes an outlet port for releasing conducting liquidfrom the channel.

The microfluidic device depicted in FIG. 2 also includes outlets to oneor more print heads and to a waste container or channel.

As discussed above, some embodiments, such as those described inconnection with FIG. 1, include affixing one or more discrete entities101 to a substrate 108. Substrate 108 includes a surface, e.g., asurface 109, upon which a layer of fluid, e.g., substrate fluid 110,e.g., oil, may be provided or deposited. Suitable substrate fluids mayinclude, for example, one or more liquids in which discrete entities areinsoluble and/or immiscible, such as water and/or oil depending on thenature of the discrete entities. Substrate fluids may be the same typeof fluid as a carrier fluid, e.g., a fluid having the same compositionas a carrier fluid, e.g., a fluid including water and/or oil, or may bea different type of fluid than the carrier fluid, e.g., a fluidincluding water and/or oil.

In some embodiments, the disclosed methods may include moving one ormore discrete entities through a device and/or affixing one or morediscrete entities to a substrate and/or removing the discrete entitiesfrom the substrate by changing the buoyancy of the discrete entitiesand/or exerting one or more forces on one or more components, e.g.,beads, of the discrete entities. Embodiments of the methods also includereleasing one or more discrete entities, e.g., an affixed discreteentity, from a substrate by, for example, modulating, e.g., modulatingby removing, one or more force affixing the entity to the substrate. Insome instances, discrete entities are removed from a substrate byremoving an electric field affixing them thereto.

To facilitate the above manipulations, the present disclosure provides,in some embodiments, a substrate which includes an array of individuallycontrollable electrodes. Such substrates may be configured such thatindividual electrodes in the array can be selectively activated anddeactivated, e.g., by applying or removing a voltage or current to theselected electrode. In this manner, a specific discrete entity affixedvia a force applied by the electrode may be selectively released from asubstrate surface, while unselected discrete entities remain affixed viaapplication of the force. The electrodes of such an array may beembedded in a substrate material (e.g., a suitable polymer material),e.g., beneath a surface of the substrate to which the discrete entitiesare affixed via application of the force. A variety of suitableconductive materials are known in the art which may be utilized inconnection with the disclosed electrode arrays, including variousmetals. Liquid electrodes as described previously herein may also beused for such an application.

Methods and devices for affixing discrete entities to a substrate arenow described. One embodiment of affixing a discrete entity, e.g., adroplet 601, generated in a microfluidic device, to a substrate 602, byapplying a force, e.g., a dielectrophoretic force, is shown in FIG. 6,panels A- C. FIG. 6, panel A, shows a droplet 601 being ejected from adelivery orifice of a microfluidic nozzle 603 of a microfluidic deviceand prior to affixation to a substrate 602. Imbedded electrodes features604 are patterned beneath the surface of substrate 602. FIG. 6, panel B,shows the droplet caught at a gap separating electrode features 604.Panel C illustrates the stretching of the trapped droplet via theapplication of dielectrophoretic force. Positioning of the substrate 602relative to the nozzle 603 is achieved, for example, with a computercontrolled mechanical stage. Alternatively, or in addition, the nozzle603 may be provided as part of a print head, e.g., a computer controlledprint head, which is movable relative to substrate 602.

The subject methods also include methods of adding reagents to adiscrete entity, e.g., a droplet, e.g., a droplet affixed to asubstrate. Such methods may include delivering and/or affixing a firstdiscrete entity in a first carrier fluid to a substrate or a portionthereof, e.g., a substrate surface. The methods may also includedelivering one or more other discrete entities, e.g., a second droplet,such as a discrete entity in a second carrier fluid and/or including oneor more reagents, to a location on the substrate which is the samelocation as the first discrete entity or a location adjacent or inproximity to that of the first discrete entity. The first and subsequentapplied discrete entities may then be coalesced such that the contents,including, for example, one or more reagents, of the first andsubsequent discrete entities are combined. In some embodiments,coalescence is spontaneous and in other embodiments, coalescing discreteentities includes applying a force, such as an electrical force, to oneor more of the discrete entities. For example, applying an electricalfield to two or more droplets in close proximity can induce dynamicinstability at the oil-water interfaces that results in droplet mergerto reduce the surface energy of the oil-water system. The first andsecond carrier fluids, as described above, may be the same type of fluidor different types of fluids.

Some embodiments of this disclosure also include methods of adding oneor more reagent and/or components, such as one or more beads, to one ormore discrete entities, e.g., droplets, by delivering one or morediscrete entities in a first carrier fluid via a first orifice of adevice to a surface of a substrate. The methods may also includepositioning one or more of such discrete entities in a second carrierfluid, e.g., a carrier fluid which is of the same or a different typethan the first carrier fluid, on the substrate surface and/or affixingthe discrete entities to the substrate via a force. According to thesubject methods, an orifice of a device, such as an orifice operablyconnected, e.g., fluidically connected, to a reagent source, may then beinserted into one or more of the affixed discrete entities. Uponinsertion, the orifice may be utilized to inject one or more reagentsinto the one or more discrete entities.

Embodiments of the methods may include modulating the environment of adiscrete entity and thereby modulating the contents of the discreteentity, e.g., by adding and/or removing contents of the droplet. Suchmodulation may include modulating a temperature, pH, pressure, chemicalcomposition, and/or radiation level of an environment of one or morediscrete entities. Such modulation may also be of the immediateenvironment of one or more discrete entities, such as an emulsion inwhich the discrete entities are provided and/or one or more space, suchas a conduit, channel, or container, within a microfluidic device. Animmediate environment of a discrete entity which may be modulated mayalso include a fluid volume, such as a fluid flow, in which the discreteentity is provided. One or more discrete entities may also be stored ina modulated environment.

The methods of this disclosure may also include recovering all or aportion of one or more discrete entities which have been affixed to asubstrate. For example, one or more materials, such as one or moresolvents and/or reagents may be recovered from a droplet via, forexample, extraction. Such a recovery may be conducted by contacting oneor more affixed discrete entities with a portion of a device, such as amicrofluidic orifice connected to a suction device for sucking one ormore material, such as one or more solvent and/or reagent from one ormore affixed discrete entity. A microfluidic orifice may be insertedinto a discrete entity and/or placed in proximity to a discrete entity,e.g., placed at a distance from a discrete entity having an order ofmagnitude of a discrete entity or smaller, for performing recovery fromthe entity. Embodiments of the methods of recovery from a discreteentity may also include shearing, e.g., detaching, a discrete entityfrom a substrate surface by, e.g., increasing the buoyancy of one ormore discrete entities. The buoyancy of a discrete entity can beincreased by increasing the volume of the discrete entity by, forexample, injecting aqueous fluid or non-aqueous fluid into the discreteentity.

In some embodiments, the methods may include concentrating one or morecomponents, e.g., beads, present in a discrete entity at a locationwithin a discrete entity. Such concentrated components or alternatively,portions of the discrete entity not containing the components, may thenbe selectively removed, e.g., removed by suction, from the discreteentity. One or more components removed from discrete entities may thenbe conveyed into one or more isolated containers via, for example, adelivery orifice.

In various aspects, substrates for use in connection with the disclosedmethods include one or more channels filled with one or more conductive,e.g., electrically conductive, liquid or solid materials, e.g., anelectrode material. In some embodiments, such substrates may alsoinclude an insulating sheet positioned between the channels and thecarrier fluid. In some embodiments, one or more channels are configured,e.g., patterned, to generate an electric field above a portion of asubstrate, such as an insulating sheet, upon application of a voltage tothe one or more channels. In some embodiments, such a voltage and aresulting electrical field or an aspect thereof, e.g., adielectrophoretic force, is sufficient to affix one or more discreteentities to the substrate. In some embodiments, a substrate, or aportion thereof, includes one or more electrodes having a net chargewhich is opposite in polarity, e.g., negative or positive, relative tothe polarity of one or more discrete entities, e.g., droplets, beingaffixed to the substrate.

As shown in FIG. 1, in some embodiments, surfaces of substrates includeone or more electrodes 111. In various embodiments, one or moreelectrodes are pre-formed on a substrate or portion thereof, e.g., asubstrate surface. Substrates may, in various embodiments, be mountedupon and/or adjacently to, e.g., contacting, a stage, such as a movablestage, such as a stage movable in an X-Y and/or Z direction. In someembodiments, a stage is movable in a direction toward and/or or awayfrom a microfluidic device, or a portion thereof, e.g., a deliveryorifice 107. Also, in some embodiments, a microfluidic device, or aportion thereof, e.g., a delivery orifice 107 is movable in a directiontoward and/or or away from another portion of a device, e.g., a stage,and/or a substrate. A stage and/or a microfluidic device, or a portionthereof, e.g., a delivery orifice, may be movable in constant movementor in increments on a scale of a diameter or radius of one or morediscrete entities, e.g., 5 or less, 10 or less, 50 or less, or 100 orless discrete entities. A stage and/or a microfluidic device, or aportion thereof, may be movable in one or more direction, e.g., an Xand/or Y and/or Z direction, in one or more increments having a distanceof, for example, 1 μm to 1000 μm, inclusive, such as 1.0 μm to 750 μm,10 μm to 500 μm, 1 μm to 50 μm, or 1 μm to 10 μm, inclusive. In someembodiments, the devices may me movable in constant movement or one ormore increments on a scale to correspond with positions on a substratewhere discrete entities may be attached, such as wells on a well plateincluding any of the well plates described herein.

In some embodiments, the methods include affixing one or more discreteentities 101 to a substrate 108, or a portion thereof, e.g., a surface109, via wetting, e.g., electrowetting. In some embodiments, wettingincludes moving, e.g., flowing, one or more discrete entities 106 from adelivery orifice 107, through a substrate fluid 110, to a substratesurface 109 of a substrate. In some embodiments, the wettability of asubstrate is sufficient to attach one or more discrete entities to thesubstrate via, for example, wetting forces. In some embodiments, themethods include modifying, e.g., increasing or decreasing, thewettability of a substrate so as to be sufficient to affix a discreteentity to the substrate via wetting forces. Various aspects of themethods may also include applying exogenous electromagnetic radiation inan amount sufficient to affix a discrete entity to a specific locationon a substrate.

In some embodiments, the subject methods include patterning one or morechannels, e.g., channels of a substrate or aspects thereof, to provide aplurality of charged electrode features in a grid pattern. Such anarrangement is shown, for example, in FIG. 4, which depicts a substrate401, including electrode features 402 and 403. A nozzle includingdelivery orifice 406 is also shown. Droplets are affixed to the gridpattern using dielectrophoresis, which allows the application of forcesto uncharged conductive droplets suspended in a nonconductive medium.For example, in one embodiment, unaffixed droplets 408 experience a netforce towards the regions on the surface of the substrate with thehighest electric field gradient, the gap 405 between oppositely chargedfeatures 402 and 403. Once droplets are brought to the substratesurface, the surfactant layer stabilizing the droplets is disrupted, andthe droplet wets the region 405. Due to the abrupt changes in geometry,the highest electric field gradients occur at the boundaries betweencharged features 402 and 403 and the gap 405. Droplets wetting theregion 405 experience a lateral force towards 402 and 403, which causesa flattening and elongation of the droplet that is proportionate to theapplied electric field. The charged features 402 and 403, do notnecessarily need to have constant and opposite polarities. For example,high electric field gradients in 405 can be created by electrifyingfeature 402 with a high voltage AC signal (1.5 kV, 30 kHZ) whilegrounding feature 403. As long as feature 403 is an adequate conductor,an ungrounded feature 403 will experience charge reorganization as aresult of an applied AC signal on 402, and will provide a functionsimilar to grounding this feature. This effect is known aselectromagnetic shielding. The patterned substrate depicted in FIG. 4may be fabricated using standard microfluidics techniques. For example,a molded PDMS device may be placed with microfluidic channels facing upand bonded to a thin polymer film. After punching, the channels may befilled with a saltwater solution and attached to a power supply, whereone network of channels becomes feature 402 and the other network ofchannels becomes feature 403.

As illustrated in FIG. 1, affixing one or more discrete entities, e.g.,discrete entities 101, to a substrate, e.g., a substrate 108, or aportion thereof, e.g., a surface 109, may include attaching the discreteentities to the substrate, e.g., substrate 108, via a force, such as agravitational, electrical, and/or magnetic force. As such, in someembodiments, a delivery orifice, e.g., a delivery orifice 107, ispositioned above a substrate 108. In some embodiments, the methodsinclude applying an electrical voltage and/or current to electrodes,e.g., electrodes 111, positioned in or on the substrate, e.g., substrate108. Affixing one or more discrete entities, e.g., discrete entities101, to a substrate, e.g., a substrate 108, or a portion thereof, mayalso include affixing the entities to the substrate via interfacialtension.

Methods of affixing are also shown in FIG. 5 wherein discrete entities502 are delivered to a patterned substrate 501 via an orifice, e.g., adelivery orifice 503, of a microfluidic device. FIG. 5 also illustratesunfilled positions 504 on a substrate 501 where discrete entities may beaffixed, e.g., affixed by a force, such as a dielectrophoretic force, tothe substrate.

Embodiments of the disclosed methods, for example the disclosed methodsas described with reference to FIG. 1, may also include a step or stepsof storing one or more discrete entities, e.g., one or more discreteentities 106 which are affixed to a substrate, e.g., a substrate 108, ora portion thereof, e.g., a surface 109. Methods of storing the discreteentities may include maintaining one or more affixed entities undercontrolled environmental conditions, e.g., at a fixed temperature and/orpressure, for a storage period. In some embodiments, one or more forcesare applied and/or maintained to maintain the one or more affixedentities in an affixed state for the entire storage period.

In some embodiments of the disclosed methods, one or more microfluidicdevices are integrated with an automated system which selectivelypositions one or more portions of the microfluidic devices, e.g., one ormore delivery orifices, relative to a substrate or a portion thereof,e.g., a substrate surface. Accordingly, in some embodiments the methodsinclude selectively positioning, e.g., positioning at a particularlocation using an automated system, one or more delivery orificesrelative to a substrate or a portion of a substrate to selectivelydeliver one or more discrete entities to one or more locations on or inproximity to the substrate or a portion thereof, e.g., a substratesurface. Automated systems as disclosed may include one or more controlunits, e.g., control units including a central processing unit, tocontrol one or more aspects of applying discrete entities to asubstrate, such as physical positioning of one or more delivery orificeand/or timing of discrete entity dispensing. Automated systems may beconfigured to position, e.g., position independently, one or moredelivery orifices with respect to a stationary substrate or position asubstrate with respect to one or more stationary delivery orifices.Aspects of the subject methods may include delivering a first member ofa plurality of discrete entities to a first location on or in proximityto a substrate or a portion thereof, e.g., a substrate surface, and asecond member of the plurality of discrete entities to the firstlocation or a second location on or in proximity to the substrate.

The subject methods may also include modulating, e.g., changing one ormore aspect of, one or more force, e.g., by modulating an electric fieldand/or buoyancy of a discrete entity in one or more carrier solution, tothereby move one or more discrete entities, e.g., a droplet, from afirst affixed location on a substrate to another location. The methodsmay also include applying one or more additional, e.g., second, forcewhich is sufficient to move one or more discrete entities from a firstaffixed location to a second location on a substrate and/or affix theone or more discrete entities at the second location. Aspects of themethods may also include applying a cross flow of fluid and/or exogenouselectromagnetic radiation sufficient to move a discrete entity from afirst location, e.g., a first affixed location, on a substrate to asecond location on a substrate.

Embodiments of the subject methods may also include performing one ormore assays, e.g., one or more biological assays, such as any of theassays described herein, on and/or in one or more of the discreteentities before and/or after delivery of a discrete entity to asubstrate or a portion thereof, e.g., a substrate surface. In someembodiments, such substrates may include a well plate or a portionthereof. The term “well plate”, is used broadly herein, to refer to aplate having one or more wells, e.g., divots or compartments, therein,such as a mictrotiter plate. However, as used herein, the term “wellplate” may also refer to a patterned array of discrete entities, e.g.,droplets, as described herein, which discrete entities are affixed to asubstrate surface. In such embodiments, the substrate surface mayinclude traditional wells, such as divots or compartments, but mayalternatively be a flat surface.

Standard assays employ well plates having, for example, a 384 wellformat, such as the well plate shown in FIG. 7, panel A. However, wellplates which may be prepared and/or utilized in accordance with thesubject methods and devices, e.g., well plates including ordered arraysof discrete entities, may include well plates having, for example, from20,000 to 500,000, inclusive, wells, such as from 50,000 to 150,000,inclusive, such as from 80,000 to 120,000, inclusive, such as 100,000wells. Such a well plate, which may have the same size footprint as thewell plate of FIG. 7, panel A, is illustrated, for example, by FIG. 7,panel B (e.g., a 128 mm×85 mm footprint). In such a well plate, eachwell may have an area ranging, for example, from 0.01 mm² to 1 mm²,inclusive, such as from 0.05 mm² to 0.5 mm², such as about 0.10 mm².Additionally, FIG. 7, panel C, illustrates magnified top and side viewsof a portion of the well plate shown in FIG. 7, panel B. FIG. 7, panelC, specifically illustrates an oil layer on a substrate having aqueousdroplets affixed thereon.

Furthermore, FIG. 7, panel D, provides a graph demonstrating how thesubject methods, for example using picodrop printing, can increase thearray density using a standard well plate footprint. Accordingly, themethods described herein enable a significantly increased array densitywithin a standard well plate footprint allowing for the performance of asignificantly increased number of assays and/or experiments. Suchmethods allow, for example, the performance of assays on a number ofsamples that is significantly higher than is achievable in a set amountof time and/or using a set amount of space according to standardmethods.

Aspects of the disclosed methods may also include controlling, e.g.,maintaining, the temperature of one or more discrete entities beforeand/or after delivery of the one or more entities to a substrate or aportion thereof, e.g., a substrate surface. For example, in someembodiments, one or more discrete entities are thermalcycled beforeand/or after delivery to a substrate or a portion thereof, e.g., asubstrate surface.

The subject methods may also include printing a structure, e.g., athree-dimensional structure, by employing a device, such as the devicedepicted generally in FIG. 1. In some embodiments, the methods includedirecting a first layer and/or a second layer and/or one or moreadditional layers, e.g., 3 to 1000 layers, inclusive, such as 10 to 500or 50 to 100 layers, of discrete entities, e.g., droplets, to asubstrate or a portion thereof, e.g., a substrate surface. In someinstances, a substrate, or a portion thereof, e.g., a substrate surface,includes thereon a layer of aqueous fluid which is miscible with acarrier fluid and immiscible with the fluid of the discrete entities,and wherein the droplets are affixed to a surface of a substrate, e.g.,a substrate surface, following their introduction into the layer ofaqueous fluid. In various embodiments, the discrete entities include oneor more solid and/or gel materials, such as one or more polymers.Aspects of the disclosed methods may also include initiating and/orsustaining a reaction, e.g., a photopolymerization reaction, whichcauses discrete entities and/or a carrier fluid of discrete entities tosolidify, e.g., solidify on a substrate to which the discrete entitiesare applied.

Exemplary Embodiments

Exemplary, non-limiting embodiments of the present disclosure areprovided below. While these are described with respect to droplets,droplet “printing”, and related devices and systems, it should beunderstood that such embodiments may be equally applicable to theprinting of non-droplet discrete entities as well.

In one embodiment of the present disclosure, an emulsion includingdroplets of different composition is “printed” to a substrate using amicrofluidic print head, e.g., as described herein. The droplets aremade ahead of time using a microfluidic or non-microfluidic technique,such as flow focusing or membrane emulsification, respectively. Thepre-formed droplets are then introduced into the print head and sortedon demand according to their fluorescence. The droplet solutions aredyed with different solutions prior to being encapsulated as droplets sothat, when injected into the print head, a detection technique, such asflow dropometry, can be used to identify each droplet's type and, usingthis information, a computer can determine which droplets to sort.

This allows dispensing of precise solutions to the substrate. Oncedispensed by the print head, the droplets are affixed to the substrateusing a force such as, for example, a dielectrophoretic force that isgenerated via electrodes fabricated under the substrate surface. A layerof oil above the substrate allows the droplets to remain in the carrierfluid at all times, that is, the droplets are in the carrier fluid aftergeneration, flowed via carrier fluid throughout the print head, and thendispensed into a carrier-fluid coated substrate. This keeps the dropletsencapsulated at all times and protects against evaporation. In additionto dielectrophoresis, other forces can also be applied to affix thedroplets. For example, an electrical force can be applied in which thesubstrate can be charged oppositely to the droplets, creating anelectrical attraction. The droplets can be charged as they pass throughthe microfluidic print head using a channel comprising charged fluidthat contacts the droplets or, for example, a salt water electrode asdescribed herein. Other forces that can be used are, for example,gravitational, in which the density of the droplets being larger thanthat of the carrier fluid causes them to sink into a well patterned onthe substrate or float into a an upside-down well, if less dense thanthe carrier fluid. Magnetic forces can be used in similar ways. Wettingand chemical forces can also be used such that the droplets, uponcontacting the substrate, wet the surface and are adhered to it viasurface tension.

In addition to a suitably designed “emulsion ink” including droplets ofdifferent type labeled with detection components that make each typedistinguishable, a “sorting on demand” microfluidic device for directingspecific droplets to the substrate at controlled times, and a substrateconstructed so as to maintain droplets at specific locations, a systemwhich automates positioning of the substrate under the dispensing nozzlewith the sorting on demand device helps provide for high-speed targeteddispensing of droplets. This can be accomplished using, for example,electrically-controlled microscope stages to position the substrateunder the nozzle, and a computer to detect and sort droplets on demandand in registry with the substrate.

The droplet dispenser is, in essence, a highly miniaturized andextremely high throughput liquid handling robot and, as such, it isvaluable for performing a variety of applications, particularlybiological assays. For example, droplets comprising reagents, cells, andother components, can be dispensed to the substrate, subjected tochanging environmental conditions, such as heating for incubation, andmonitored over time to measure reaction activity. The results obtainedfrom monitoring the system can be integrated together with thedispensing platform to, for example, change the conditions in specificdroplets by adding additional reagents based on reaction progress.

In some embodiments of the present disclosure, the described system canbe used to “print” cells and tissues. In such embodiments, the cells ortissue building blocks are first encapsulated in droplets labeled withdetection components along with necessary biological reagents, such asmatrigel or collagen. The resulting emulsion ink, which can containcells of different types, cell aggregates, or biological reagentswithout cells, can then be sorted on demand via the print head anddispensed to the substrate, where they are affixed with a force. Tolocalize cells on the array, traps can be positioned with space betweenthem. To print tissues, the cells are preferably deposited sufficientlyclose so as to allow neighboring droplets to coalesce and the cellscontained within them to interact with one another. This can be used,for example, to print a “red” cell next to a “green” cell next to a“blue” cell. These steps can be repeated to make a line of cells in adesired pattern. Additional lines can then be printed adjacent to thefirst line to print a flat layer of cells. Additional layers can then beprinted above the first layer, to generate a 3D multi-layered “tissue”.With proper selection or engineering of the cells, once the tissue isprinted, the cells can interact with one another to further modify thestructure. Additional droplets can be added to modulate the structuresdevelopment, such as biological reagents or drugs.

In similar embodiments, cell aggregates of defined type can be localizedon the array in separate droplets. For example, a first position on thearray can be dispensed with a specific combination of cells, such as ared, then a green, and then a blue cell. These cells will be dispensedinto the same droplet by droplet addition in which they can theninteract to perform functions. This can be repeated at additional spotson the array to build multiple identical aggregates or different,defined aggregates. This can be used, for example, to build elementarytissue structures composed of just tens of thousands of cells, or tostudy interactions between different cell types such as bacterial andmammalian cells, or microbes with infecting virus. Drugs and otherchemical and biological compounds can also be added to the droplets, forexample, to study how to modulate the interactions between theorganisms.

In some embodiments the described system can be used to analyze cells.In some such embodiments, cells are isolated in the droplets on thearray and additional materials are added as needed. The materials canstimulate the cells to grow, express defined pathways associatedproteins, or include a microbe or virus that can infect the cells. Usinga detection technique, such as optical detection, fluorescence, Ramanmicroscopy, or other spectrographic techniques that preserve thedroplets and the cells within them, it would be possible to monitor thedroplets over time. This could be used, for example, to detect a changein a specific reaction or time-dependent expression of a target pathway.Using other techniques, including destructive techniques like massspectrometry, PCR, or sequencing, with or without molecular barcodes, itwould be possible to detect molecular information.

In some embodiments, cell-free experiments can be performed in thearrayed droplets. For example, cell-free extracts such as transcriptionand translation machinery can be encapsulated in the droplets, alongwith other components, including, if desired, cells. These can then beincubated on the array and monitored over time, as described above, totrack progress of the reaction. This can be used, for instance, toscreen pathways for activity in cell-free extracts and to investigatehow pathway activity is modulated with changing conditions, such as theapplication of heat or presence of different inducers, inhibitors, etc.

Synthetic biology screening: The methods described herein can be used toperform screens for synthetic biology applications such as, for example,screening cells or cell-free extracts engineered to express biologicalpathways that produce molecules. By isolating the pathways in dropletson the array and tracking the production of the molecules using methodslike microscopy, spectroscopy, or mass spectrometry, it is possible totest different pathway sequences for desired activity.

Mass spectrometry activated sorting: In another embodiment, thedescribed system can be used to sort droplets or cells using a mass-specread out. For example, droplets containing different materials or cellscan be dispensed to the array. A portion of each droplet can then besampled and introduced into a mass spectrometry, to analyze itscontents. Based on the information obtained, all or a portion of thedroplet can be recovered for additional use.

3D printing with materials: In another embodiment of the disclosedmethods, discrete materials comprising solids, liquids, or solidifiablematerials can be printed to the substrate, to generate planar or “3D”structures. For example, solid particles can be generated using avariety of processes, such as emulsion polymerization or droplet-basedtemplating in which the material is emulsified into droplets whileliquid and then solidified to convert a liquid droplet into a solidparticle of similar dimensions. An “ink” comprising these solidparticles can be generated by mixing together multiple particles ofdifferent type with different labels that can be determined optically.The particle-based ink can then be introduced into the print head andsorted on demand to the substrate, thereby depositing solid particles tothe substrate in the desired pattern. Trapping forces like electrostaticor magnetic forces can also be used to localize the particles at definedpositions. A first layer of particles can be deposited, and afterwards,additional layers can be added, to generate 3D structures in which thecomposition of each particle in the structure is defined exactly. Oncedeposited, a variety of methods can be used to bond the particlestogether such as, for example, chemical bonding techniques or sinteringof the particles. In a slightly different embodiment, the aforementioned“3D printer” can print liquid droplets that can be solidified afterbeing dispensed using, for example, chemical cross linking,polymerization, or gelation. The materials that are printed can comprisehydrophilic or hydrophobic liquids, metals, and plastics, with thecarrier fluid and forces being selected as needed to enable controlledsorting on demand and dispensing of the materials to the substrate.

Sorting on demand: In microfluidic and other applications it is oftendesirable to generate droplets of defined type on demand. One method foraccomplishing this is using a microfluidic droplet generator controlledby a membrane valve. When the valve is closed, the dispersed phase doesnot flow and no droplets are generated. When it is opened, it flows anddroplets are generated. This approach can generate droplets on demand asfast as the valve can opened and closed, which is often no faster than100 Hz. In addition, the droplets are all formed of the same fluid; toenable generation of droplets on demand from multiple fluids, multipledevices, each with its own fluid, may be interfaced together; this ischallenging for more than a handful of fluids. Such a challenge may beaddressed by embodiments of the present disclosure wherein droplets aregenerated on demand by sorting them, from a preexisting emulsion, ondemand. The droplets of the different desired fluids are firstemulsified separately and combined into a single mixed emulsion. Theyare labeled to enable them to be differentiated from one another usingoptical detection, such as flow cytometry. This combined emulsion isthen injected into a microfluidic sorter which scans the droplets andsorts them down two channels, a dispensing channel and a waste channel.From the perspective of the dispensing channel, this system includes adroplet on demand technique since, by diverting droplets down thedispensing channel on demand, droplets are ejected from the channel ondemand. The emulsion that is sent into waste can be recycled through thesorter, to conserve reagents. The value of this droplet on demandtechnique is that it is limited in speed to the rate at which thedroplets can be sorted. With new sorting geometries incorporating gappeddividers between the sorting outlets (as described in greater detailherein), it is possible to sort droplets at >30 kHz, which is more thantwo orders of magnitude faster than can be achieved with publisheddroplet on demand techniques. In addition, the combined emulsion cancontain droplets of many different types, not just tens of droplets buthundreds or thousands of droplets. This allows sequences of droplets ofunprecedented complexity to be generated, which is important inconnection with the described printing technology for allowingcontrolled dispensing and combining of different reagents at eachsubstrate location.

The sorting on demand device provides the control which facilitatesdispensing defined sequences of droplets, but the trapping substrateallows for the capture those droplets at specific locations so that oneor more assays of interest can be performed on them. There are a varietyof substrates that can be constructed for trapping the droplets. Onesuch substrate uses dielectrophoresis to trap the droplets. To generatethe dielectrophoretic traps, the substrate may be fabricated so as tocontain electrodes with which to generate the requisite electric fields.This can be accomplished by patterning electrodes under a dielectricsheet; the electrodes can be energized with positive and negativecharges to generate large electric fields with a spatial gradient; whendroplets are dispensed above the substrate and in the region of thefield, dielectrophoretic forces will cause them to be attracted to thesubstrate, and adhere. The electrodes can be patterned usingconventional fabrication techniques, such as metal sputtering ordeposition on the sheet, or by fabricating microfluidic channels thatcan be bonded face-side-up to the bottom side of the sheet such that thechannels are below the sheet and not in fluidic communication with thefluids above the sheet. The channels can then be filled with conductivemedium, such as solder or electrolyte solution and charged to generatethe desired electric fields for dielectrophoretic droplet tapping. Bymodulating the shapes, widths, and heights of the microfluidic channels,it is possible to structure the electrodes, thereby providing controlover the fields that are applied to the droplets above the sheet.

Affixing droplets: In some embodiments, it is desirable to affix liquidor solid entities to the surface of a substrate via application of aforce. One such force that can be used is dielectrophoresis, in which apatterned array of electrodes under the dielectric substrate is used togenerate electric fields that dielectrophoretically attract or repel theentities, trapping them at the desired locations.

Non-dielectrophoretic electrical forces can also be used. In suchembodiments, the entities can be charged with, for example, a positivecharge, either before, during, or after their flow through the printhead. The substrate can then be charged oppositely, creating anelectrical attraction between the entities and the substrate that willaffix them. The polarity of the entities and substrate can also bemodulated to generate a repulsive force allowing, for example, dropletsto be ejected from the substrate. Electrodes can be used for thesepurposes. For example, the substrate can be uniformly charged with onepolarity so that droplets of the opposite polarity will stick to thesubstrate. Provided a dielectric separates the electrode from thedroplets, no charge will flow between the two and the force will remain;alternatively, if the two are allowed to come into electrical contact,then charge will flow, removing the force but allowing, for example, adroplet to wet to the electrode and be affixed by interfacial tensionforces.

In another embodiment, the electrodes can be patterned so that each traphas a single or multiple electrodes with the same or different polarityand charge. This can be used, for example, to generate dielectrophoretictraps appropriate to affix single droplets. Each electrode can beaddressable and a large array of the traps can be fabricated into thesubstrate, allowing each drop to be switched on or off as desired. Thiscan be used, for example, to capture droplets to specific traps bymodulating the strength of the field of the trap where the droplet is tobe affixed relative to other traps in the vicinity. The traps can alsobe turned off, to selectively release drops.

Different affixing forces are also possible, such as wettability andinterfacial tension forces. In such embodiments, the substrate can bepatterned with regions that alternate between hydrophilic andhydrophobic. For example, the substrate can be natively hydrophobic butpatterned with small islands large enough to accommodate one or multipledrops with hydrophilic wettability. The wettability patterning can beaccomplished with, for example, spatially-modulated light-based polymergrafting or flow patterning of polyelectrolyte layers. Once the dropletsare in contact with the hydrophilic patch, they may wet spontaneously orthey may be induced to wet, for instance if surfactants are present, byapplying a small, transient or long-lived electric field. Once wetted tothe substrate, the droplets can be maintained for periods of time.

A method to trap droplets, which utilizes interfacial tension, may beaccomplished with patterned features. For example, wells can befabricated into the substrate and sized and/or shaped such that dropletsfit therein and, due to buoyancy or density differences with the carrierfluid, sit within the wells. The droplets can also be dispensed within aconcave feature with a narrow opening, or between posts with narrowgaps. Such droplets may be held in place due to their interfacialtension and preference for remaining spherical.

Other kinds of electromagnetic traps can be generated using, forexample, laser tweezers. Using an array of lasers directed at controlledlocations on the substrate, droplets dispensed near the lasers mayexperience a force attracting or repelling them to or from the lasers,again generating a series of traps that can be used to localize thedroplets. Magnetic droplets or particles can be affixed using magneticor electromagnetic forces such as, for example, with ferrofluids,permanent magnets, paramagnetism, or electromagnetism generated byflowing electric current through an electrode patterned under thesubstrate.

Modulating the position of droplets: Once droplets are dispensed to thesubstrate, it is possible to change the position of the droplets on thesubstrate. This can be accomplished, e.g., magnetically, by modulatingthe magnetic field, electrically or dielectrophoretically, by modulatingelectric fields, via electrowetting on dielectric, or by varying theposition of optical traps with the lasers, among other forces. Thecarrier fluid, e.g., oil, surrounding the droplets can also be flowed soas to apply a shear to the droplets affixed to the substrate, causingthem in some instances to move in the direction of the flow. If thedroplets have a different density from the carrier fluid, buoyancy canalso be used to move the droplets by altering the orientation of thesubstrate in a gravitational field.

Adding reagents to droplets: In some embodiments, it may be desirable todispense multiple droplets or discrete entities to a single location onthe substrate array. This is valuable, for example, for adding differentreagents to localized droplets at different and defined times. This canbe accomplished by, for example, dispensing a first droplet to the arrayand then dispensing a second droplet to the same position as the firstdroplet. In certain embodiments, such as when electric fields are usedto trap the drops, the electric fields generated by the substrate aresufficient to induce the droplets to merge, thereby combining theircontents. The contents of the droplets can be mixed via diffusion orconvective flow in the droplets generated by, for example, convection ofthe carrier fluid over the surface of the droplet or motion of thedroplet when the trap is moved. In other instances, droplets will mergespontaneously, such as when no surfactants are used. In other instances,merger can be induced via application of a laser or localized heating.Additional drops can be added to the same location to add, one, two,three, or more droplets to the same position. Using the droplet orsorting on demand techniques, the drops that are added and the sequencein which they are added can be controlled exactly. In anotherembodiment, a nozzle or capillary can be introduced into the affixeddroplet to inject the desired reagent.

Recovering droplets or material therefrom: In certain applications, itis desirable to recover all or portions of the affixed droplets. Thiscan be accomplished, for example, by bringing a nozzle close to theaffixed droplet and drawing fluid into the nozzle, thereby drawing thedroplet into the nozzle. Alternatively, the nozzle can be used togenerate a localized flow of carrier fluid which can be used to dispeldroplets from the surface by overcoming the affixing force. If thenozzle shape is designed appropriately and the fluid flow adequate, itis also possible to recover a portion of the droplet in a mechanismsimilar to microcapillary-based droplet generation. Alternatively, or inaddition, droplets can be removed from the substrate by addingadditional liquid to them to increase their buoyancy; once the buoyantforce is larger than the affixing force, the droplet will detach fromthe substrate and float away. If the droplets are heavier than thecarrier, a similar result can be accomplished by inverting thesubstrate. In embodiments in which the traps can be selectively switchedon and off, droplets can also be recovered by switching off the forceand using buoyancy or flow to remove them from the substrate and recoverthem into a collection container.

Concentrating materials in droplets: In some embodiments, it isdesirable to concentrate reagents or other materials in the droplets.This can be accomplished using available techniques for concentratingreagents such as, for example, placing beads in the droplets that canbind certain components in the droplets, and then either removing thebead or the portion of the droplet that does not contain the bead toachieve a concentration increase.

Secondary manipulation of droplets: The portions or complete dropletsrecovered with any of the methods described herein can then be dispensedinto a secondary container by flowing them from the array into thecontainer. For example, using the section method, individual droplets ordroplet portions can be recovered from the droplet array and theseportions flowed through a tube into a well on a well plate, where theyare dispensed. This can be done one droplet at a time, dispensing eachdroplet into a separate well and thereby preserving the isolation of thedroplets from one another. Once in the well, other operations can beperfumed on the droplet, such as propagating cells contained therein orperforming biological reactions, such as ELISA, PCR, etc. Molecularanalysis using techniques like microscopy, spectroscopy, and massspectrometry can also be performed on the recovered entities.

Manipulating affixed droplets: Affixed droplets can be manipulated usinga variety of techniques to modulate their environment. For example, insome embodiments, it is possible to modulate the chemical, temperature,or pressure environment of the droplets to perform, for example, PCR bythermocycling the droplets. The carrier fluid can also be replaced tomodulate the chemical properties of the droplet and to transportmaterials in and out of the droplets. For example, carrier phases withsurfactant may undergo micellar transport, allowing compounds to betransported into or out of the droplets without merging dropletstogether. This can be used, for example, to stain compounds, e.g.,oil-based compounds, with dyes that have an affinity for oil, e.g., nilered, by saturating the carrier with nile red and flowing it over thedroplets for a period. In some embodiments, once the droplets areaffixed, surfactants may no longer be necessary since the droplets neednot be in contact; the traps can be spaced as necessary to accomplishthis. In this scenario, the micellar transport can be reduced byreplacing the oil with an oil that contains no surfactant. This could bevaluable for enhancing the containment of compounds in the droplets thatotherwise would leak out due to micellar transport. Similarly, thedroplets can be dispersed using a carrier phase that is optimized forthis step, and then the carrier replaced with a different carrier thathas other desirable properties, such as the ability to enhance or reducethe partitioning of reagents out of or into the droplets. These kinds ofenvironmental manipulations can be used to prepare the droplets forlonger-term storage, such as at low temperature, to preserve reagentswithin them.

Alternatively, or in addition, the substrate can be fabricated to have asemi-permeable membrane that allows chemical communication with thedroplets from below the substrate (or above depending on the orientationof the substrate relative to the droplets). By modulating the fluidsunder the membrane, chemical partitioning can be used to modulate thecontents of the droplets while still preserving the droplets intact. Forexample, this can be used to change the buffering properties of thedroplets by dispensing the droplets with a first buffer, e.g.,containing ions, and then using the membrane, placing the droplets inchemical communication with another buffer with different ions, allowingthe ions in the droplets to be replaced with those from the new buffersolution. By controlling the permeability of the membrane, the types ofcompounds that are modulated can be controlled based on, for example,their size, hydrophobic, charge, or chemical properties.

Analyzing affixed droplets: In some applications of the invention, it isdesirable to analyze the contents of the affixed droplet. For example,this is valuable for monitoring the change of a detectable marker, suchas a fluorescent signal resulting from, e.g., PCR amplification or thefluorescent product of an enzymatic substrate used in an ELISA. In theseinstances, the droplets may be analyzed in a similar way to which thevolumes of a well plate are analyzed with a plate-reader. They can bemonitored over time, to collect information of how these signals varyover time. This can be used, for example, to screen a pathway thatproduces a peak concentration in product as a function of time, in whichthe peak height and width are both important parameters for optimizationof the pathway, or to perform quantitative PCR in the droplets onnucleic acids or cells.

Multiple measurement modalities can be used, such as bright field,fluorescence, and absorbance techniques. Spectrographic techniques canalso be applied, such as Raman spectroscopy, NMR, and mass spectrometry.Separation techniques can also be used, such as capillaryelectrophoresis by making contact with the droplets through, forexample, a nozzle or capillary. By recovering all or portions of thedroplets, the material can also be subjected to destructive ornon-destructive techniques, such as mass-spectrometry and chemicalanalysis. Importantly, since the nozzle position is known during thematerial recovery process, the signals and information recovered fromthese and other assays can be traced back to specific droplets on thearray, allowing time-resolved information to be combined with powerfulmolecular analysis techniques, such as sequencing of the material in thedroplet portions.

Parallel print heads: A single print head is limited in the rate atwhich it can dispense droplets to the substrate. One technique fordispensing droplets more quickly is to parallelize the print heads. Inthis approach, multiple nozzles can be attached to a single droplet ondemand device and/or sorter on demand device so that, when a droplet istriggered, it is split into multiple portions, each of which isdispensed to the substrate at a different, defined location. This canalso be used to dispense groups of droplets that were known to originatefrom the same parent droplet, and are thus related in certain ways. Dueto the modular nature of the print heads, it is also possible toassemble multiple print heads together into a single device. Forexample, the use of the fiber optics for the detection of the dropletsallows the detection optics to be localized to a small region in thedevice, while the sorting or droplet on demand devices, themselves, areonly hundreds of microns in total size. This allows multiple devices tobe assembled on a single chip so as to dispense droplets out of a singlecombined nozzle, or multiple outlet nozzles. In theory, this shouldallow printing at rates increased by a factor equal to the number ofdevices assembled on the print head.

Tagging droplets with a unique identifier: In certain applications it isvaluable to tag the contents of the printed droplets with a uniqueidentifier. This allows for the contents of multiple droplets to bepooled together while keeping track of from which droplet each entityoriginated. One such example of this is nucleic acid tagging, orbarcoding. In this approach, for example, single cells can be localizedin the droplets, lysed, and their nucleic acids tagged with uniqueidentifiers relating from which droplet, and thus, from which cell (inthe case of single cell encapsulation), each nucleic acid originated.The nucleic acids can then all be pooled and sequenced and the tags usedto group them according to single droplets and cells. Other examples inwhich this would be valuable would be the tagging of different segmentsof a viral genome or the amplification, fragmentation, and tagging theportions of a long DNA molecule, allowing, in essence, long sequencereads to be generated from short, tagged reads.

Multiple fibers for detection: To analyze the fluorescence of a droplet,it is necessary to provide excitation light, e.g., in the form of alaser, and read the generated emission light. In some embodiments of theinvention, this can be accomplished using a single optical fiber thatserves both to funnel the excitation light into the device and alsocollects the emitted light in the reverse direction. A drawback of thisapproach, however, is that the optical properties that are ideal forexcitation light guidance may not be the same as for emission lightcapture. For example, to excite a narrow beam, a fiber with a narrow tipis preferred, but to collect the largest number of emitted photons, awide fiber with a large collecting cone angle is preferred. In theseinstances, multiple fibers can be used. For example, a narrow fiber canbe used to provide a concentrated, excitation signal, while a wide fibercan collect the emitted fluorescent light.

qPCR in printed droplets: In some embodiments, the methods, devices,and/or systems described herein can be used to quantitate nucleic acids.In this approach, sample droplets comprising nucleic acids are dispensedto the substrate. Reagents necessary for amplification are also added tothe droplets, either by combining them with the sample droplets prior todispensing, or by dispensing additional droplets to the positions of thesample containing droplets, wherein the additional droplets include thenecessary reagents and a detection component, where the detectioncomponent signals the amplification. The droplet are then incubatedunder conditions suitable for amplification and monitored to read thedetection component. This provides, for each droplet, a rate of changeof the detection component which can be used to quantitate the nucleicacids in the droplets. In addition to quantitating DNA and RNA, thisapproach can also be used to quantitate the nucleic acids within livingorganisms, including viruses and single cells. In such embodiments,additional steps suitable for efficient cell lysis, such as theinclusion of lysis buffers, may be implemented using droplet addition.

Single cell barcoding: In some embodiments, the methods, devices, and/orsystems described herein can be used to sequence single cells. Forexample, individual cells can be encapsulated in the droplets anddispensed to the substrate as described herein. The cells can then belysed and subjected to molecular biological processing to amplify and/ortag their nucleic acids with barcodes. The material from all thedroplets can then be pooled for all cells and sequenced and the barcodesused to sort the sequences according to single droplets or cells. Thesemethods can be used, for example, to sequence the genomes ortranscriptomes of single cells in a massively parallel format.Alternatively, the cells, prior to encapsulation, can be bound withantibodies that are themselves labeled with tags relating the type ofanybody they are. For example, a cocktail of tens or thousands ofantibodies, each labeled with a tag relating the type of antibody it is,can be used to stain a collection of cells. The cells can then bedispensed and subjected to a barcoding protocol where the tags on theantibodies are additionally labeled with a tag/barcode relating thedroplet/cell it originated from. This protocol is similar to the singlecell sequencing protocol except that rather than labeling nucleic acidsoriginating in the cell, it labels ones carried with the cell by theantibodies. The approach can be used to detect surface proteins in, forexample, living or dead cells, or internal proteins with, for example, afixation and permeabilization protocol. Similar techniques can beapplied to individual viruses, macromolecular complexes, and proteins.

Synthesizing polymers: The ability to deliver droplets of definedcomposition to specific locations on a substrate is valuable for polymersynthesis. For example, in one embodiment, a first droplet can bedispensed to the substrate surface which includes a first monomer orpolymer. A second droplet can then be dispensed to the same or anadjacent location, which includes a second monomer or polymer. The firstand second droplets can then be incubated under and/or exposed toconditions sufficient for the contents of the first and second dropletsto come into contact and for the first polymer or first monomer to forma covalent bond with the second polymer or second monomer, therebygenerating a synthesized polymer. These steps can be repeated toincrease the length of the polymer and thereby create polymers ofdefined sequence. In alternative embodiments, techniques like GibsonAssembly can be used for nucleic acid synthesis, which allows for theassembly of multiple components added at the same time, where overlapsequences are used to control the order in which the pieces are linkedto synthesize a polymer of defined sequence. This can be used, forexample to build DNA constructs for synthetic biology applications.

Screening libraries: Droplet based microfluidic techniques are valuablefor screening libraries of compounds, enzymes, cells, etc., in which (orin connection with which) a reaction occurs that, normally, cannot beconfined. For example, in directed evolution of enzymes, the product ofa successful enzymatic reaction is a molecule that, generally, diffusesaway from the enzyme catalyst. If many enzymes of varying catalyticpower exist within the same solution, the product molecules mix,preventing the molecules produced by the action of one enzyme from beingidentified as having been produced by that enzyme. To evolve an enzyme,it is important to be able to select the best variant in a population(or a variant having a desired enzymatic activity relative to the othermembers of the population), which requires a method for measuring enzymeactivity through product concentration. By enclosing each enzyme in adifferent droplet, it is possible to measure the activity of eachvariant independently by measuring the product concentration in eachdroplet. This can also be performed in the printed droplet format. Forexample, each enzyme variant can be localized in a droplet on the arrayand assayed for activity, and efficient enzymes (or those having adesired enzymatic activity) can be obtained by recovering theencapsulating droplets. Similar screens can be performed to test fortherapeutic efficacy of a drug, e.g., a small molecule drug, or drugcombination by evaluating its effects on cells in the droplets.Alternatively, by observing the droplets over time, it is also possibleto screen based on time-dependent measurements, such as a peakproduction in product concentration at a specific time and/or for aspecific duration.

Printing microarrays: In some embodiments, the methods, devices, and/orsystems described herein can be used to synthesis oligos on an array formicroarray production. For example, the substrate can be functionalizedwith a moiety to which nucleic acids can be attached. Then, bysequentially dispensing droplets of specific nucleic acids to individualspots on the substrate surface, the sequences can be attached to thesubstrate. The resolution of the spots will depend on the resolutionwith which the droplets can be printed, which is the on the order ofmicro to nanoscale features.

In situ sequencing: In some embodiments, the methods, devices, and/orsystems described herein can be used for in situ sequencing. In thisapproach, the goal is to correlate sequence information with the spatiallocation of the nucleic acids in the system, such as a nucleic acidsoriginating within a cell in a tissue or tumor. This can be accomplishedby printing onto such tissues droplets containing tags that relate thelocation of the tag. For example, the top left corner of a tissue can beprinted with a tag that relates that the coordinate of the tag is at thetop left corner, where different tag sequences, for example comprisingnucleic acids, can be used for different coordinates on the tissue. Thetags can be allowed to diffuse into the tissue, and a slice of thetissue can then be removed and disaggregated into small portions, suchas single cells. This can then be repeated for the next slice, using tagsequences that relate the 3D position of each portion in the tissue. Theportions, once disaggregated, can then be subjected to the single cellbarcoding approach described above, where, in addition to sequences suchas the genomic DNA and transcriptome RNA, the tags relating the locationof that portion of material in the original sample are also barcoded.These materials can then be sequenced providing the sequences of thenucleic acids in the biological system and the location, by way of thetag sequence, that the portion originated from. This embodiment wouldallow, for example, the full genomic, transcriptional and proteomicinformation for every cell in the system to be obtained with aresolution equal to the droplet printing resolution and the slicingthickness.

Types of Discrete Entities

The composition and nature of the discrete entities, e.g.,microdroplets, prepared and or utilized in connection with the disclosedmethods may vary. For example, in some embodiments, a discrete entitymay include one cell and not more than once cell. In other embodiments,a discrete entity may include a plurality of cells, i.e., two or morecells. In some aspects, discrete entities according to the presentdisclosure may include a nucleic acid or a plurality of nucleic acids.In some embodiments, as discussed above, discrete entities may includeone or more solid and/or gel materials, such as one or more polymers.

In some embodiments, a surfactant may be used to stabilize the discreteentities, e.g., microdroplets. Accordingly, a microdroplet may involve asurfactant stabilized emulsion. Any convenient surfactant that allowsfor the desired reactions to be performed in the discrete entities,e.g., microdroplets, may be used. In other aspects, a discrete entity,e.g., a microdroplet, is not stabilized by surfactants or particles.

The surfactant used depends on a number of factors such as the oil andaqueous phases (or other suitable immiscible phases, e.g., any suitablehydrophobic and hydrophilic phases) used for the emulsions. For example,when using aqueous droplets in a fluorocarbon oil, the surfactant mayhave a hydrophilic block (PEG-PPO) and a hydrophobic fluorinated block(Krytox® FSH). If, however, the oil was switched to be a hydrocarbonoil, for example, the surfactant would instead be chosen so that it hada hydrophobic hydrocarbon block, like the surfactant ABIL EM90. Inselecting a surfactant, desirable properties that may be considered inchoosing the surfactant may include one or more of the following: (1)the surfactant has low viscosity; (2) the surfactant is immiscible withthe polymer used to construct the device, and thus it doesn't swell thedevice; (3) biocompatibility; (4) the assay reagents are not soluble inthe surfactant; (5) the surfactant exhibits favorable gas solubility, inthat it allows gases to come in and out; (6) the surfactant has aboiling point higher than the temperature used for PCR (e.g., 95° C.);(7) the emulsion stability; (8) that the surfactant stabilizes drops ofthe desired size; (9) that the surfactant is soluble in the carrierphase and not in the droplet phase; (10) that the surfactant has limitedfluorescence properties; and (11) that the surfactant remains soluble inthe carrier phase over a range of temperatures.

Other surfactants can also be envisioned, including ionic surfactants.Other additives can also be included in the oil to stabilize thediscrete entities, e.g., microdroplets, including polymers that increasediscrete entity, e.g., droplet, stability at temperatures above 35° C.

The discrete entities, e.g., microdroplets, described herein may beprepared as emulsions, e.g., as an aqueous phase fluid dispersed in animmiscible phase carrier fluid (e.g., a fluorocarbon oil or ahydrocarbon oil) or vice versa. The nature of the microfluidic channel(or a coating thereon), e.g., hydrophilic or hydrophobic, may beselected so as to be compatible with the type of emulsion being utilizedat a particular point in a microfluidic work flow.

Emulsions may be generated using microfluidic devices as described ingreater detail below. Microfluidic devices can form emulsions consistingof droplets that are extremely uniform in size. The microdropletgeneration process may be accomplished by pumping two immiscible fluids,such as oil and water, into a junction. The junction shape, fluidproperties (viscosity, interfacial tension, etc.), and flow ratesinfluence the properties of the microdroplets generated but, for arelatively wide range of properties, microdroplets of controlled,uniform size can be generated using methods like T-junctions and flowfocusing. To vary microdroplet size, the flow rates of the immiscibleliquids may be varied since, for T-junction and flow focus methodologiesover a certain range of properties, microdroplet size depends on totalflow rate and the ratio of the two fluid flow rates. To generate anemulsion with microfluidic methods, the two fluids are normally loadedinto two inlet reservoirs (syringes, pressure tubes) and thenpressurized as needed to generate the desired flow rates (using syringepumps, pressure regulators, gravity, etc.). This pumps the fluidsthrough the device at the desired flow rates, thus generatingmicrodroplet of the desired size and rate.

Adding Reagents to Discrete Entities

In practicing the subject methods, a number of reagents may be added to,i.e., incorporated into and/or encapsulated by, the discrete entities,e.g., microdroplets, in one or more steps (e.g., about 2, about 3, about4, or about 5 or more steps). Such reagents may include, for example,amplification reagents, such as Polymerase Chain Reaction (PCR)reagents. The methods of adding reagents to the discrete entities, e.g.,microdroplets, may vary in a number of ways. Approaches of interestinclude, but are not limited to, those described by Ahn, et al., Appl.Phys. Lett. 88, 264105 (2006); Priest, et al., Appl. Phys. Lett. 89,134101 (2006); Abate, et al., PNAS, Nov. 9, 2010 vol. 107 no. 4519163-19166; and Song, et al., Anal. Chem., 2006, 78 (14), pp 4839-4849;the disclosures of which are incorporated herein by reference.

For instance, a reagent may be added to a discrete entity, e.g.,microdroplet, by a method involving merging a discrete entity, e.g., amicrodroplet, with a second discrete entity, e.g., microdroplet, whichcontains the reagent(s). The reagent(s) that are contained in the seconddiscrete entity may be added by any convenient methods, specificallyincluding those described herein. This second discrete entity may bemerged with the first discrete entity to create a discrete entity, e.g.,a microdroplet, which includes the contents of both the first discreteentity and the second discrete entity.

One or more reagents may also, or instead, be added using techniquessuch as droplet coalescence, or picoinjection. In droplet coalescence, atarget drop (i.e., the microdroplet) may be flowed alongside amicrodroplet containing the reagent(s) to be added to the microdroplet.The two microdroplets may be flowed such that they are in contact witheach other, but not touching other microdroplets. These drops may thenbe passed through electrodes or other aspects for applying an electricalfield, wherein the electric field may destabilize the microdroplets suchthat they are merged together.

Reagents may also, or instead, be added using picoinjection. In thisapproach, a target drop (i.e., the microdroplet) may be flowed past achannel containing the reagent(s) to be added, wherein the reagent(s)are at an elevated pressure. Due to the presence of the surfactants,however, in the absence of an electric field, the microdroplet will flowpast without being injected, because surfactants coating themicrodroplet may prevent the fluid(s) from entering. However, if anelectric field is applied to the microdroplet as it passes the injector,fluid containing the reagent(s) will be injected into the microdroplet.The amount of reagent added to the microdroplet may be controlled byseveral different parameters, such as by adjusting the injectionpressure and the velocity of the flowing drops, by switching theelectric field on and off, and the like.

In various aspects, one or more reagents may also, or instead, be addedto a microdroplet by a method that does not rely on merging two dropletstogether or on injecting liquid into a drop. Rather, one or morereagents may be added to a microdroplet by a method involving the stepsof emulsifying a reagent into a stream of very small drops, and mergingthese small drops with a target microdroplet. Such methods shall bereferred to herein as “reagent addition through multiple-dropcoalescence.” These methods take advantage of the fact that due to thesmall size of the drops to be added compared to that of the targetdrops, the small drops will flow faster than the target drops andcollect behind them. The collection can then be merged by, for example,applying an electric field. This approach can also, or instead, be usedto add multiple reagents to a microdroplet by using several co-flowingstreams of small drops of different fluids. To enable effective mergerof the tiny and target drops, it is important to make the tiny dropssmaller than the channel containing the target drops, and also to makethe distance between the channel injecting the target drops from theelectrodes applying the electric field sufficiently long so as to givethe tiny drops time to “catch up” to the target drops. If this channelis too short, not all tiny drops will merge with the target drop, addingless reagent than desired. To a certain degree, this can be compensatedfor by increasing the magnitude of the electric field, which tends toallow drops that are farther apart to merge. In addition to making thetiny drops on the same microfluidic device, they can also, or instead,be made offline using another microfluidic drop maker or throughhomogenization and then injecting them into the device containing thetarget drops.

Accordingly, in some embodiments a reagent is added to a microdroplet bya method involving emulsifying the reagent into a stream of droplets,wherein the droplets are smaller than the size of the microdroplet;flowing the droplets together with the microdroplet; and merging adroplet with the microdroplet. The diameter of the droplets contained inthe stream of droplets may vary ranging from about 75% or less than thatof the diameter of the microdroplet, e.g., the diameter of the flowingdroplets is about 75% or less than that of the diameter of themicrodroplet, about 50% or less than that of the diameter of themicrodroplet, about 25% or less than that of the diameter of themicrodroplet, about 15% or less than that of the diameter of themicrodroplet, about 10% or less than that of the diameter of themicrodroplet, about 5% or less than that of the diameter of themicrodroplet, or about 2% or less than that of the diameter of themicrodroplet. In certain aspects, a plurality of flowing droplets may bemerged with the microdroplet, such as 2 or more droplets, 3 or more, 4or more, or 5 or more. Such merging may be achieved in a variety ofways, including but not limited to by applying an electric field,wherein the electric field is effective to merge the flowing dropletwith the microdroplet.

A reagent, in another aspect, is added to a drop (e.g., a microdroplet)formed at an earlier time by enveloping the drop to which the reagent isbe added (i.e., the “target drop”) inside a drop containing the reagentto be added (the “target reagent”). In certain embodiments such a methodis carried out by first encapsulating the target drop in a shell of asuitable hydrophobic phase, e.g., oil, to form a double emulsion. Thedouble emulsion is then encapsulated by a drop containing the targetreagent to form a triple emulsion. To combine the target drop with thedrop containing the target reagent, the double emulsion is then burstopen using any suitable method, including, but not limited to, applyingan electric field, adding chemicals that destabilizes the dropletinterface, flowing the triple emulsion through constrictions and othermicrofluidic geometries, applying mechanical agitation or ultrasound,increasing or reducing temperature, or by encapsulating magneticparticles in the drops that can rupture the double emulsion interfacewhen pulled by a magnetic field.

Sorting

In practicing the methods of the present disclosure, one or more sortingsteps may be employed. Sorting approaches of interest include, by arenot necessarily limited to, approaches that involve the use of one ormore sorters, e.g., sorters of a microfluidic device, which employmicrofluidic valves, membrane valves, bifurcating channels, surfaceacoustic waves, and/or dielectrophoresis. Sorting approaches which maybe utilized in connection with the disclosed methods, systems anddevices also include those depicted in FIG. 2, and those described byAgresti, et al., PNAS vol. 107, no 9, 4004-4009; the disclosure of whichis incorporated herein by reference. A population, e.g., a population ofdiscrete entities, may be enriched by sorting, in that a populationcontaining a mix of members having or not having a desired property maybe enriched by removing those members that do not have the desiredproperty, thereby producing an enriched population having the desiredproperty.

In various embodiments, the subject methods include scanning, e.g.,optically scanning one or more discrete entities, e.g., microdroplets,to facilitate sorting of the discrete entities. As such, in someembodiments, microfluidic devices or portions thereof, e.g., sorters,include one or more detectors, e.g., optical scanners. A variety ofsuitable optical scanners are known in the art. Such optical scannersmay include, e.g., one or more optical fibers for applying excitationenergy to one or more discrete entities. In some embodiments, a suitableoptical scanner utilizes a laser light source directed into the back ofan objective, and focused onto a microfluidic channel through whichdroplets flow, e.g., to excite fluorescent dyes within one or morediscrete entities. Scanning one more discrete entities may allow one ormore properties, e.g., size, shape, composition, of the scanned entitiesto be determined. Sorting may, in turn, be carried out based on the oneor more properties. For example, sorting may be based on resultsobtained from an optical scan of one or more discrete entities.

Properties of discrete entities which may be detected include, but arenot limited to, the size, viscosity, mass, buoyancy, surface tension,electrical conductivity, charge, magnetism, and/or presence or absenceof one or more components, e.g., one or more detectable labels (e.g.,one or more fluorescent labels). In certain aspects, sorting may bebased at least in part upon the presence or absence of one or more cellsin the microdroplet, e.g., one or more detectably labeled cells. Incertain aspects, sorting may be based at least in part based upon thedetection of the presence or absence of PCR amplification products.

Sorting may be applied at any suitable point in the disclosed methods.Moreover, two or more sorting steps may be applied to a population ofdiscrete entities or types thereof, e.g., microdroplets, e.g., about 2or more sorting steps, about 3 or more, about 4 or more, or about 5 ormore, etc. When a plurality of sorting steps is applied, the steps maybe substantially identical or different in one or more ways (e.g.,sorting based upon a different property, sorting using a differenttechnique, and the like).

Moreover, discrete entities, e.g., droplets, may be purified prior to,or after, any sorting step. In one embodiment a droplet may be purifiedas follows: a majority of the fluid in the drop is replaced it with apurified solution, without removing any discrete reagents that may beencapsulated in the drop, such a cells or beads. The microdroplet isfirst injected with a solution to dilute any impurities within it. Thediluted microdroplet is then flowed through a microfluidic channel onwhich an electric field is being applied using electrodes. Due to thedielectrophoretic forces generated by the field, as the cells or otherdiscrete reagents pass through the field they will be displaced in theflow. The drops are then split, so that all the objects end up in onemicrodroplet. Accordingly, the initial microdroplet has been purified,in that the contaminants may be removed while the presence and/orconcentration of discrete reagents, such as beads or cells, which may beencapsulated within the droplet, are maintained in the resultingmicrodroplet.

Microdroplets may be sorted based on one or more properties. Propertiesof interest include, but are not limited to, the size, viscosity, mass,buoyancy, surface tension, electrical conductivity, charge, magnetism,and/or presence or absence of one or more components, e.g., one or moredetectable labels. In certain aspects, sorting may be based at least inpart upon the presence or absence of one or more cells in themicrodroplet, e.g., one or more detectably labeled cells. In certainaspects, sorting may be based at least in part based upon the detectionof the presence or absence of PCR amplification products.

Sorting may be employed, for example, to remove discrete entities, e.g.,microdroplets, in which no cells are present. Encapsulation may resultin one or more discrete entities, e.g., microdroplets, including amajority of the discrete entities, e.g., microdroplets, in which no cellis present. If such empty drops were left in the system, they would beprocessed as any other drop, during which reagents and time would bewasted. To achieve the highest speed and efficiency, these empty dropsmay be removed with droplet sorting. For example, a drop maker mayoperate close to the dripping-to-jetting transition such that, in theabsence of a cell, drops of a first size, e.g., 8 μm, are formed; bycontrast, when a cell is present the disturbance created in the flowwill trigger the breakup of the jet, forming drops of a second size,e.g., 25 μm in diameter. The device may thus produce a bi-dispersepopulation of empty drops of a first size, e.g., 8 μm, and single-cellcontaining drops of a second size, e.g., 25 μm, which may then be sortedby size using, e.g., a hydrodynamic sorter to recover only the,single-cell containing drops of the second, e.g., larger, size.

Sorters of the subject embodiments may be active or passive sorters.Passive sorters of interest include hydrodynamic sorters, which sortdiscrete entities, e.g., microdroplets, into different channelsaccording to size, based on the different ways in which small and largedrops travel through the microfluidic channels. Also of interest arebulk sorters, a simple example of which is a tube containing drops ofdifferent mass in a gravitational field. By centrifuging, agitating,and/or shaking the tube, lighter drops that are more buoyant willnaturally migrate to the top of the container. Drops that have magneticproperties could be sorted in a similar process, except by applying amagnetic field to the container, towards which drops with magneticproperties will naturally migrate according to the magnitude of thoseproperties. A passive sorter as used in the subject methods may alsoinvolve relatively large channels that will sort large numbers of dropssimultaneously based on their flow properties. Additionally, in someembodiments, sorting is carried out via activation of one or morevalves, e.g., microfluidic valves.

Picoinjection can also be used to change the electrical properties ofthe drops. This could be used, for example, to change the conductivityof the drops by adding ions, which could then be used to sort them, forexample, using dielectrophoresis. Alternatively, picoinjection can alsobe used to charge the drops. This could be achieved by injecting a fluidinto the drops that is charged, so that after injection, the drops wouldbe charged. This would produce a collection of drops in which some werecharged and others not, and the charged drops could then be extracted byflowing them through a region of electric field, which will deflect thembased on their charge amount. By injecting different amounts of liquidby modulating the piocoinjection, or by modulating the voltage to injectdifferent charges for affixed injection volume, the final charge on thedrops could be adjusted, to produce drops with different charge. Thesewould then be deflected by different amounts in the electric fieldregion, allowing them to be sorted into different containers.

Improved Sorting Architecture for High-Speed Sorting of Microdroplets

In some embodiments, the present disclosure provides microfluidicdevices with an improved sorting architecture, which facilitates thehigh-speed sorting of discrete entities, e.g., microdroplets. Thissorting architecture may be used in connection with the microdropletprinter embodiments described herein or in any other suitableapplication where high-speed sorting of microdroplets is desired.Related methods and systems are also described. For example, in someembodiments, microfluidic devices are provided which include at least aninlet channel; a first outlet channel in fluid communication with theinlet channel; a second outlet channel in fluid communication with theinlet channel; a dividing wall separating the first outlet channel fromthe second outlet channel, wherein the dividing wall comprises a firstproximal portion having a height which is less than the height of theinlet channel and a second distal portion having a height which is equalto or greater than the height of the inlet channel.

In some embodiments, the height of the first proximal portion of thedividing wall is from about 10% to about 90% of the height of the inletchannel, e.g., from about 20% to about 80%, from about 30% to about 70%,from about 40% to about 60%, or about 50% of the height of the inletchannel.

In some embodiments the height of the first proximal portion of thedividing wall is from about 10% to about 20%, from about 20% to about30%, from about 30% to about 40%, from about 40% to about 50%, fromabout 50% to about 60%, from about 60% to about 70%, or from about 80%to about 90% of the height of the inlet channel.

In some embodiments, the length of the proximal portion of the dividingwall is equal to or greater than the diameter of a microdroplet asdescribed herein, e.g., a microdroplet to be sorted using a microfluidicdevice as described herein. For example, in some embodiments, the lengthof the proximal portion of the dividing wall is from about 1× to about100× the diameter of a microdroplet as described herein, e.g., fromabout 1× to about 10×, from about 10× to about 20×, from about 20× toabout 30×, from about 30× to about 40×, from about 40× to about 50×,from about 50× to about 60×, from about 60× to about 70×, from about 70×to about 80×, from about 80× to about 90×, or from about 90× to about100× the diameter of a microdroplet as described herein.

In some embodiments a microfluidic device according to the presentdisclosure includes an electrode, e.g., a liquid electrode, configuredto selectively apply an electrical field in an inlet channel of themicrofluidic device upstream of the dividing wall to effect sorting ofone or more microdroplets.

In some embodiments, liquid electrodes include liquid electrode channelsfilled with a conducting liquid (e.g. salt water or buffer) and situatedat positions in the microfluidic device where an electric field isdesired. In particular embodiments, the liquid electrodes are energizedusing a power supply or high voltage amplifier. In some embodiments, theliquid electrode channel includes an inlet port so that a conductingliquid can be added to the liquid electrode channel. Such conductingliquid may be added to the liquid electrode channel, for example, byconnecting a tube filled with the liquid to the inlet port and applyingpressure. In particular embodiments, the liquid electrode channel alsoincludes an outlet port for releasing conducting liquid from thechannel.

In alternative embodiments, a solid electrode prepared from any suitableconductive material may be utilized.

As described herein, microfluidic devices according to the presentdisclosure may include a moat salt solution (to generate the fieldgradient used for dielectrophoretic deflection and to limit stray fieldsthat can cause unintended droplet merger) provided in suitable channels.

Accordingly, a microfluidic device having a gapped dividing wall isprovided which facilitates high speed sorting as described in greaterdetail in the Experimental section. The gapped dividing wall of thepresent disclosure in combination with one or more detectors asdescribed herein, and one or more electrodes as described hereinfacilitate the high-speed sorting of microdroplets.

Printing Cell Layers

In some embodiments, the present disclosure provides methods and relateddevices and systems for printing one or more tissues and/or cell layers.FIG. 3 depicts a non-limiting, simplified representation of one type ofmicrofluidic system and method of the present disclosure which may beutilized in the printing of one or more tissues or cell layers. FIG. 3illustrates the delivery of discrete entities including cells to asubstrate. In one such method, discrete entities, e.g., droplets 301,are prepared using a device, e.g., a microfluidic device 300, and acarrier fluid 302 to produce a mixed emulsion including the discreteentities. Discrete entities, e.g., droplets 301, as shown in FIG. 3 mayinclude one or more reagent, e.g., a reagent which facilitates cellgrowth and/or a cell culture media component and/or a cell culturesubstrate, e.g., a matrigel, and different types of cells 303. Thesubject method may include encapsulating, e.g., encapsulating by fullycontaining therein, one or more cells in a discrete entity.

In some aspects of printing tissues, a mixed emulsion is flowed througha sorter 304 which sorts discrete entities 301 of the mixed emulsionbased on one or more of their characteristics. As is shown in FIG. 3,the sorter 304 may be configured to detect and/or separate discreteentities 301 containing cells 303 based on cell type. For example, asorter may be configured to separate a first type of discrete entity,e.g., a droplet type containing one or more cell of interest, and asecond type, e.g., a type not containing one or more cell of interest.As such, a sorter 304 may produce a first fraction, e.g., a fluidcontaining carrier fluid and discrete entities having a first type,e.g., one or more type of interest, and a second fraction, e.g., a fluidcontaining carrier fluid and discrete entities having a second type,e.g., one or more type not of interest. The sorter 304 may also beconfigured to direct the first fraction toward a portion of amicrofluidic device having a delivery orifice 305, e.g., a nozzle havinga delivery orifice 305, for delivery to a substrate and the secondfraction toward a waste container or outlet. Alternatively, the secondfraction may be recycled by reintroducing it upstream of the sorter 504.In some embodiments, a nozzle or a portion thereof, e.g., a deliveryorifice 305, may be positioned from about 1 μm to about 200 μm, such asfrom about 5 μm to about 100 μm, or from about 10 μm to about 50 μm,inclusive, such as about 20 μm away from a target such as surface 306 ofa substrate or a previously deposited layer of discrete entities.

In some variations, the methods may include affixing a first layer 307of discrete entities, e.g., discrete entities including a first celltype, to a substrate surface 306 of a substrate and one or more otherlayers, e.g., a second layer 308 of discrete entities, e.g., discreteentities including a second cell type, to the first layer of discreteentities. In various embodiments, a first layer 307 of discrete entitiescan be applied to a substrate surface 306 before, or contemporaneouslywith, a second layer 308. Aspects of the methods may also includeaffixing one or more additional layers of discrete entities, e.g.,discrete entities encapsulating cells, to one or more previously affixedlayer. For example, the subject methods may include affixing between 1and 10 million, inclusive, such as between 10 and 1 million or between100 and 10,000, inclusive, additional layers of discrete entities.Accordingly, the methods may include providing a layered structure,e.g., a tissue, by repeated layering of discrete entity layers. Itshould be noted that each layer may include discrete entities of aspecific type or a plurality of discrete entities of different types,e.g., discrete entities having varying compositions or components. Forexample, first layer 307 and/or a second layer 308 may include aplurality of discrete entities including different cell types, e.g., afirst discrete entity including a first cell type and a second discreteentity including a second different cell type than the first discreteentity. In other words, each layer may include either a homogenous orheterogeneous population of discrete entities, e.g., microdroplets.

In some embodiments, substrates or portions thereof, e.g., substratesurfaces, include one or more electrodes 309. Such electrodes 309 may beused to apply a force, to thereby cause a first layer 307, e.g., initiallayer, of discrete entities to affix to, e.g., wet, a substrate or asubstrate surface 306 thereof. Once a first layer 307, e.g., initiallayer, of discrete entities is applied, electric field gradients at dropsurfaces of discrete entities of the first layer 307 may causesubsequent discrete entities, e.g., discrete entities of a second layer308, to affix to, e.g., wet, the first layer 307.

Detecting Cells

In some embodiments, the subject methods involve detecting the presenceand/or absence of one or more cells or one or more othercharacteristics, such as type and/or size, of one or more subset ofcells (e.g., tumor cells) in one or more discrete entities, e.g.,droplets, while in a mixed emulsion and/or before, during or after thediscrete entity is affixed to a layer of discrete entities, a substrate,or a portion thereof, e.g., a substrate surface, as described herein. Insome embodiments, a sorter of a microfluidic device is utilized fordetecting one or more characteristics of cells encapsulated withindiscrete entities.

Aspects of the disclosed methods may include detecting one or morecharacteristics of cells, e.g., one or more cells within discreteentities affixed to a substrate, at a plurality of time points, e.g., aplurality of equally-spaced time points. The methods may also includedetecting one or more characteristics of one or more cells continuouslyover a period of time, such as detecting a component of the one or morecells, and/or a product of the one or more cells. Embodiments of themethods may further include recovering, e.g., recovering by extracting,from a discrete entity one or more cells, a component of one or morecells, e.g., deoxyribonucleic acid (DNA), and/or a product of one ormore cells. In various embodiments, the methods may include sequencingDNA recovered from one or more cells.

Aspects of the disclosed methods may include incorporating into a mixedemulsion discrete entities having one or more cells obtained from abiological sample.

As used herein, the term “biological sample” encompasses a variety ofsample types obtained from a variety of sources, which sample typescontain biological material. For example, the term includes biologicalsamples obtained from a mammalian subject, e.g., a human subject, andbiological samples obtained from a food, water, or other environmentalsource, etc. The definition encompasses blood and other liquid samplesof biological origin, as well as solid tissue samples such as a biopsyspecimen or tissue cultures or cells derived therefrom and the progenythereof. The definition also includes samples that have been manipulatedin any way after their procurement, such as by treatment with reagents,solubilization, or enrichment for certain components, such aspolynucleotides. The term “biological sample” encompasses a clinicalsample, and also includes cells in culture, cell supernatants, celllysates, cells, serum, plasma, biological fluid, and tissue samples.“Biological sample” includes cells; biological fluids such as blood,cerebrospinal fluid, semen, saliva, and the like; bile; bone marrow;skin (e.g., skin biopsy); and antibodies obtained from an individual.

As is described more fully herein, in various aspects the subjectmethods may be used to detect a variety of components from cells, suchas cells from biological samples. Components of interest include, butare not necessarily limited to, cells (e.g., circulating cells and/orcirculating tumor cells), polynucleotides (e.g., DNA and/or RNA),polypeptides (e.g., peptides and/or proteins), and many other componentsthat may be present in a biological sample.

“Polynucleotides” or “oligonucleotides” as used herein refer to linearpolymers of nucleotide monomers, and may be used interchangeably.Polynucleotides and oligonucleotides can have any of a variety ofstructural configurations, e.g., be single stranded, double stranded, ora combination of both, as well as having higher order intra- orintermolecular secondary/tertiary structures, e.g., hairpins, loops,triple stranded regions, etc. Polynucleotides typically range in sizefrom a few monomeric units, e.g. 5-40, when they are usually referred toas “oligonucleotides,” to several thousand monomeric units. Whenever apolynucleotide or oligonucleotide is represented by a sequence ofletters (upper or lower case), such as “ATGCCTG,” it will be understoodthat the nucleotides are in 5′→3′ order from left to right and that “A”denotes deoxyadenosine, “C” denotes deoxycytidine, “G” denotesdeoxyguanosine, and “T” denotes thymidine, “I” denotes deoxyinosine, “U”denotes uridine, unless otherwise indicated or obvious from context.Unless otherwise noted the terminology and atom numbering conventionswill follow those disclosed in Strachan and Read, Human MolecularGenetics 2 (Wiley-Liss, New York, 1999).

The terms “polypeptide,” “peptide,” and “protein,” used interchangeablyherein, refer to a polymeric form of amino acids of any length. NH₂refers to the free amino group present at the amino terminus of apolypeptide. COOH refers to the free carboxyl group present at thecarboxyl terminus of a polypeptide. In keeping with standard polypeptidenomenclature, J. Biol. Chem., 243 (1969), 3552-3559 is used.

In certain aspects, methods are provided for counting and/or genotypingcells, including normal cells or tumor cells. A feature of such methodsis the use of microfluidics.

According to some embodiments of the subject methods, cells, e.g., cellsin discrete entities in an emulsion and/or affixed to a substrate, abiological sample (e.g., whole blood) may be recovered from a subjectusing any convenient method, e.g., by applying a needle and/or asyringe. The biological sample may then be processed to removecomponents other than cells using, for example, processing steps such ascentrifugation, filtration, and the like.

Each cell in the biological sample, or a subset thereof, may then beencapsulated into a discrete entity, e.g., a droplet, using amicrofluidic device. Methods and devices which may be utilized in theencapsulating of a component from a biological sample are described inPCT Publication No. WO 2014/028378, the disclosure of which isincorporated by reference herein in its entirety and for all purposes.Encapsulation approaches of interest also include, but are not limitedto, hydrodynamically-triggered drop formation and those described byLink, et al., Phys. Rev. Lett. 92, 054503 (2004), the disclosure ofwhich is incorporated herein by reference. Other methods ofencapsulating cells into droplets may also be applied. Where desired,the cells may be stained with one or more antibodies and/or probes priorto encapsulating them into drops.

One or more lysing agents may also be added to the discrete entities,e.g., droplets, containing a cell, under conditions in which the cell(s)may be caused to burst, thereby releasing their genomes. The lysingagents may be added after the cells are encapsulated into discreteentities, e.g., microdroplets. Any convenient lysing agent may beemployed, such as proteinase K or cytotoxins. In particular embodiments,cells may be co-encapsulated in drops with lysis buffer containingdetergents such as Triton X100 and/or proteinase K. The specificconditions in which the cell(s) may be caused to burst will varydepending on the specific lysing agent used. For example, if proteinaseK is incorporated as a lysing agent, the discrete entities, e.g.,droplets, may be heated to about 37-60° C. for about 20 min to lyse thecells and to allow the proteinase K to digest cellular proteins, afterwhich they may be heated to about 95° C. for about 5-10 min todeactivate the proteinase K.

In certain aspects, cell lysis may also, or instead, rely on techniquesthat do not involve addition of lysing agent. For example, lysis may beachieved by mechanical techniques that may employ various geometricfeatures to effect piercing, shearing, abrading, etc. of cells. Othertypes of mechanical breakage such as acoustic techniques may also beused. Further, thermal energy can also be used to lyse cells. Anyconvenient methods of effecting cell lysis may be employed in themethods described herein.

One or more primers may be introduced into the discrete entities, e.g.,droplets, for each of the genes, e.g., oncogenes, to be detected. Hence,in certain aspects, primers for all target genes, e.g., oncogenes, maybe present in the discrete entity, e.g., droplet, at the same time,thereby providing a multiplexed assay. The discrete entities, e.g.,droplets, may be temperature-cycled so that discrete entities, e.g.,droplets, containing cancerous cells, for example, will undergo PCR.During this time, only the primers corresponding to genes, e.g.,oncogenes, present in the genome will induce amplification, creatingmany copies of these genes, e.g., oncogenes, in the discrete entity,e.g., droplet. Detecting the presence of these PCR products may beachieved by a variety of ways, such as by using FRET, staining with anintercalating dye, or attaching them to a bead. More information on thedifferent options for such detection is also provided herein. Thediscrete entity, e.g., droplet, may be optically probed, e.g., probedusing a laser, to detect the PCR products. Optically probing thediscrete entity, e.g., droplet, may involve counting the number oftarget cells, e.g., tumor cells, present in the initial population,and/or to allow for the identification the target, e.g., oncogenes,present in each cell, e.g., tumor cell.

Aspects of the subject methods may be used to determine whether abiological sample contains particular cells of interest, e.g., tumorcells, or not. In certain aspects, the subject methods may includequantifying the number of cells of interest, e.g., tumor cells, presentin a biological sample. Quantifying the number of cells of interest,e.g., tumor cells, present in a biological sample may be based at leastin part on the number of discrete entities, e.g., droplets, in which PCRamplification products were detected. For example, discrete entities,e.g., droplets, may be produced under conditions in which the majorityof discrete entities, e.g., droplets, are expected to contain zero orone cells. Those discrete entities, e.g., droplets, that do not containany cells may be removed, using techniques described more fully herein.After performing the PCR steps outlined above, the total number ofdiscrete entities, e.g., droplets, that are detected to contain PCRproducts may be counted, so as to quantify the number of cells ofinterest, e.g., tumor cells, in the biological sample. In certainaspects, the methods may also include counting the total number ofdiscrete entities, e.g., droplets, so as to determine the fraction orpercentage of cells from the biological sample that are cells ofinterest, e.g., tumor cells.

PCR

As described above, in practicing the subject methods, a PCR-basedassay, e.g., quantitative PCR (qPCR), may be used to detect the presenceof certain genes of interest, e.g., oncogene(s), present in discreteentities or one or more components thereof, e.g., cells encapsulatedtherein. Such assays can be applied to discrete entities within amicrofluidic device or a portion thereof and/or while the discreteentities are affixed to a substrate or a portion thereof, e.g., asubstrate surface. The conditions of such PCR-based assays may includedetecting nucleic acid amplification over time and may vary in one ormore ways.

For instance, the number of PCR primers that may be added to amicrodroplet may vary. The term “primer” may refer to more than oneprimer and may refer to an oligonucleotide, whether occurring naturally,as in a purified restriction digest, or produced synthetically, which iscapable of acting as a point of initiation of synthesis along acomplementary strand when placed under conditions in which synthesis ofa primer extension product which is complementary to a nucleic acidstrand is catalyzed. Such conditions include, e.g., the presence of fourdifferent deoxyribonucleoside triphosphates and apolymerization-inducing agent such as DNA polymerase or reversetranscriptase, in a suitable buffer (“buffer” which includessubstituents which are cofactors, or which affect pH, ionic strength,etc.), and at a suitable temperature. The primer may be single-strandedfor maximum efficiency in amplification.

The complement of a nucleic acid sequence as used herein may refer to anoligonucleotide which, when aligned with the nucleic acid sequence suchthat the 5′ end of one sequence is paired with the 3′ end of the other,is in “antiparallel association.” Complementarity need not be perfect;stable duplexes may contain mismatched base pairs or unmatched bases.Duplex stability can be determined by empirically considering a numberof variables including, for example, the length of the oligonucleotide,percent concentration of cytosine and guanine bases in theoligonucleotide, ionic strength, and incidence of mismatched base pairs.

The number of PCR primers that may be added to a microdroplet may rangefrom about 1 to about 500 or more, e.g., about 2 to 100 primers, about 2to 10 primers, about 10 to 20 primers, about 20 to 30 primers, about 30to 40 primers, about 40 to 50 primers, about 50 to 60 primers, about 60to 70 primers, about 70 to 80 primers, about 80 to 90 primers, about 90to 100 primers, about 100 to 150 primers, about 150 to 200 primers,about 200 to 250 primers, about 250 to 300 primers, about 300 to 350primers, about 350 to 400 primers, about 400 to 450 primers, about 450to 500 primers, or about 500 primers or more.

Such primers may contain primers for one or more gene of interest, e.g.oncogenes. The number of primers for genes of interest that are addedmay be from about one to 500, e.g., about 1 to 10 primers, about 10 to20 primers, about 20 to 30 primers, about 30 to 40 primers, about 40 to50 primers, about 50 to 60 primers, about 60 to 70 primers, about 70 to80 primers, about 80 to 90 primers, about 90 to 100 primers, about 100to 150 primers, about 150 to 200 primers, about 200 to 250 primers,about 250 to 300 primers, about 300 to 350 primers, about 350 to 400primers, about 400 to 450 primers, about 450 to 500 primers, or about500 primers or more.

Such primers and/or reagents may be added to a discrete entity, e.g., amicrodroplet, in one step, or in more than one step. For instance, theprimers may be added in two or more steps, three or more steps, four ormore steps, or five or more steps. Regardless of whether the primers areadded in one step or in more than one step, they may be added after theaddition of a lysing agent, prior to the addition of a lysing agent, orconcomitantly with the addition of a lysing agent. When added before orafter the addition of a lysing agent, the PCR primers may be added in aseparate step from the addition of a lysing agent. In some embodiments,the discrete entity, e.g., a microdroplet, may be subjected to adilution step and/or enzyme inactivation step prior to the addition ofthe PCR reagents. Exemplary embodiments of such methods are described inPCT Publication No. WO 2014/028378, the disclosure of which isincorporated by reference herein in its entirety and for all purposes.

Once primers have been added to a discrete entity, e.g., a microdroplet,the discrete entity, e.g., a microdroplet, may be incubated underconditions allowing for PCR. The discrete entity, e.g., a microdroplet,may be incubated on the same microfluidic device as was used to add theprimer(s), or may be incubated on a separate device. In certainembodiments, incubating the discrete entity, e.g., a microdroplet, underconditions allowing for PCR amplification is performed on the samemicrofluidic device used to encapsulate the cells and/or lyse the cells.Incubating the microdroplets may take a variety of forms. In certainaspects, the drops containing the PCR mix may be flowed through achannel that incubates the droplets under conditions effective for PCR.Flowing the microdroplets through a channel may involve a channel thatsnakes over various temperature zones maintained at temperatureseffective for PCR. Such channels may, for example, cycle over two ormore temperature zones, wherein at least one zone is maintained at about65° C. and at least one zone is maintained at about 95° C. As the dropsmove through such zones, their temperature cycles, as needed for PCR.The precise number of zones, and the respective temperature of eachzone, may be determined to achieve the desired PCR amplification.

In other embodiments, incubating the microdroplets may involve the useof a “Megadroplet Array”, for example as described in PCT PublicationNo. WO 2014/028378, the disclosure of which is incorporated by referenceherein in its entirety and for all purposes. In such a device, an arrayof hundreds, thousands, or millions of traps indented into a channel(e.g., a PDMS channel) sit above a thermal system. The channel may bepressurized, thereby preventing gas from escaping. The height of themicrofluidic channel is smaller than the diameter of the discreteentities, e.g., drops, causing discrete entities to adopt a flattenedpancake shape. When a discrete entity flows over an unoccupiedindentation, it adopts a lower, more energetically favorable, radius ofcurvature, leading to a force that pulls the discrete entity entirelyinto the trap. By flowing discrete entities as a close pack, it isensured that all traps on the array are occupied. The entire device maybe thermal cycled using a heater.

In certain aspects, the heater includes a Peltier plate, heat sink, andcontrol computer. The Peltier plate allows for the heating or cooling ofthe chip above or below room temperature by controlling the appliedcurrent. To ensure controlled and reproducible temperature, a computermay monitor the temperature of the array using integrated temperatureprobes, and may adjust the applied current to heat and cool as needed. Ametallic (e.g. copper) plate allows for uniform application of heat anddissipation of excess heat during cooling cycles, enabling cooling fromabout 95° C. to about 60° C. in under about one minute.

Methods of the disclosure may also include introducing one or moreprobes to the microdroplet. As used herein with respect to nucleicacids, the term “probe” refers to a labeled oligonucleotide which formsa duplex structure with a sequence in the target nucleic acid, due tocomplementarity of at least one sequence in the probe with a sequence inthe target region. Probes of interest include, but are not limited to,TaqMan® probes (e.g., as described in Holland, P. M.; Abramson, R. D.;Watson, R.; Gelfand, D. H. (1991). “Detection of specific polymerasechain reaction product by utilizing the 5′—3′ exonuclease activity ofThermus aquaticus DNA polymerase”. PNAS, 88 (16): 7276-7280).

In some embodiments of the subject methods, an RT-PCR based assay isused to detect the presence of certain transcripts of interest, e.g.,oncogene(s), present in cells. In such embodiments, reversetranscriptase and any other reagents necessary for cDNA synthesis areadded to the discrete entity, e.g., microdroplet, in addition to thereagents used to carry out PCR described herein (collectively referredto as the “RT-PCR reagents”). The RT-PCR reagents are added to thediscrete entity, e.g., microdroplet, using any of the methods describedherein. Once reagents for RT-PCR have been added to a discrete entity,e.g., microdroplet, the microdroplet may be incubated under conditionsallowing for reverse transcription followed by conditions allowing forPCR as described herein. The microdroplet may be incubated on the samemicrofluidic device as was used to add the RT-PCR reagents, or may beincubated on a separate device. In certain embodiments, incubating themicrodroplet under conditions allowing for RT-PCR is performed on thesame microfluidic device used to encapsulate the cells and lyse thecells.

In certain embodiments, the reagents added to the microdroplet forRT-PCR or PCR further includes a fluorescent DNA probe capable ofdetecting real-time RT-PCR or PCR products. Any suitable fluorescent DNAprobe can be used including, but not limited to SYBR Green, TaqMan®,Molecular Beacons and Scorpion probes. In certain embodiments, thereagents added to the microdroplet include more than one DNA probe,e.g., two fluorescent DNA probes, three fluorescent DNA probes, or fourfluorescent DNA probes. The use of multiple fluorescent DNA probesallows for the concurrent measurement of RT-PCR or PCR products in asingle reaction.

Furthermore, examples of PCR-based assays of interest which may beemployed according to the subject embodiments, include, but are notlimited to, quantitative PCR (qPCR), quantitative fluorescent PCR(QF-PCR), multiplex fluorescent PCR (MF-PCR), real time PCR (RT-PCR),single cell PCR, PCR-RFLP/RT-PCR-RFLP, hot start PCR, nested PCR, insitu polony PCR, in situ rolling circle amplification (RCA), bridge PCR,picotiter PCR and emulsion PCR. Other suitable amplification methodsinclude the ligase chain reaction (LCR), transcription amplification,self-sustained sequence replication, selective amplification of targetpolynucleotide sequences, consensus sequence primed polymerase chainreaction (CP-PCR), arbitrarily primed polymerase chain reaction(AP-PCR), degenerate oligonucleotide-primed PCR (DOP-PCR) and nucleicacid based sequence amplification (NABSA).

Multiplexing

In various aspects of the subject methods, multiple biomarkers may bedetected and analyzed for a particular discrete entity or one or morecomponents thereof, e.g., cell(s) encapsulated therein. Biomarkersdetected may include, but are not limited to, one or more proteins,transcripts and/or genetic signatures in a cell's genome or combinationsthereof. With standard fluorescence based detection, the number ofbiomarkers that can be simultaneously interrogated may be limited to thenumber of fluorescent dyes that can be independently visualized withineach discrete entity, e.g., microdroplet. In certain embodiments, thenumber of biomarkers that can be individually detected within aparticular discrete entity, e.g., microdroplet can be increased. Forexample, this may be accomplished by segregation of dyes to differentparts of the discrete entity, e.g., microdroplet. In particularembodiments, beads (e.g. LUMINEX® beads) conjugated with dyes and probes(e.g., nucleic acid or antibody probes) may be encapsulated in thediscrete entity, e.g., microdroplet to increase the number of biomarkersanalyzed. In another embodiment, fluorescence polarization may be usedto achieve a greater number of detectable signals for differentbiomarkers for a single cell. For example, fluorescent dyes may beattached to various probes and the discrete entity, e.g., microdroplet,may be visualized under different polarization conditions. In this way,the same colored dye can be utilized to provide a signal for differentprobe targets for a single cell. The use of fixed and/or permeabilizedcells also may allow for increased levels of multiplexing. For example,labeled antibodies may be used to target protein targets localized tocellular components while labeled PCR and/or RT-PCR products are freewithin a discrete entity, e.g., microdroplet. This allows for dyes ofthe same color to be used for antibodies and for amplicons produced byRT-PCR.

Detecting PCR Products

The manner in which PCR products can be detected according to thesubject methods may vary. For example, if the goal is to count thenumber of a particular cell type, e.g., tumor cells, present in apopulation, this may be achieved by using a simple binary assay in whichSybrGreen, or any other stain and/or intercalating stain, is added toeach discrete entity, e.g., microdroplet, so that in the event acharacterizing gene, e.g., an oncogene, is present and PCR products areproduced, the discrete entity, e.g., microdroplet, will becomefluorescent. The change in fluorescence may be due to fluorescencepolarization. The detection component may include the use of anintercalating stain (e.g., SybrGreen).

A variety of different detection components may be used in practicingthe subject methods, including using one or more fluorescent dyes. Suchfluorescent dyes may be divided into families, such as fluorescein andits derivatives; rhodamine and its derivatives; cyanine and itsderivatives; coumarin and its derivatives; Cascade Blue and itsderivatives; Lucifer Yellow and its derivatives; BODIPY and itsderivatives; and the like. Exemplary fluorophores includeindocarbocyanine (C3), indodicarbocyanine (C5), Cy3, Cy3.5, Cy5, Cy5.5,Cy7, Texas Red, Pacific Blue, Oregon Green 488, Alexa fluor-355, AlexaFluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor-555, AlexaFluor 568, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 660, AlexaFluor 680, JOE, Lissamine, Rhodamine Green, BODIPY, fluoresceinisothiocyanate (FITC), carboxy-fluorescein (FAM), phycoerythrin,rhodamine, dichlororhodamine (dRhodamine), carboxy tetramethylrhodamine(TAMRA), carboxy-X-rhodamine (ROX), LIZ, VIC, NED, PET, SYBR, PicoGreen,RiboGreen, and the like. Descriptions of fluorophores and their use, canbe found in, among other places, R. Haugland, Handbook of FluorescentProbes and Research Products, 9th ed. (2002), Molecular Probes, Eugene,Oreg.; M. Schena, Microarray Analysis (2003), John Wiley & Sons,Hoboken, N.J.; Synthetic Medicinal Chemistry 2003/2004 Catalog, Berryand Associates, Ann Arbor, Mich.; G. Hermanson, Bioconjugate Techniques,Academic Press (1996); and Glen Research 2002 Catalog, Sterling, Va.

In practicing the subject methods, therefore, a component may bedetected based upon, for example, a change in fluorescence. In certainaspects, the change in fluorescence is due to fluorescence resonanceenergy transfer (FRET). In this approach, a special set of primers maybe used in which the 5′ primer has a quencher dye and the 3′ primer hasa fluorescent dye. These dyes can be arranged anywhere on the primers,either on the ends or in the middles. Because the primers arecomplementary, they will exist as duplexes in solution, so that theemission of the fluorescent dye will be quenched by the quencher dye,since they will be in close proximity to one another, causing thesolution to appear dark. After PCR, these primers will be incorporatedinto the long PCR products, and will therefore be far apart from oneanother. This will allow the fluorescent dye to emit light, causing thesolution to become fluorescent. Hence, to detect if a particular targetgene, e.g., oncogene, is present, one may measure the intensity of thediscrete entity, e.g., droplet, at the wavelength of the fluorescentdye. To detect if different target genes, e.g., oncogenes, are present,this would be done with different colored dyes for the differentprimers. This would cause the discrete entity, e.g., droplet, to becomefluorescent at all wavelengths corresponding to the primers of thetarget genes, e.g., oncogenes, present in the cell.

In some embodiments, the disclosed methods may include a step ofencapsulating or incorporating unique identifier molecules, e.g.,nucleic acid barcodes, into a plurality of discrete entities, e.g.,droplets, such that each discrete entity of the plurality of discreteentities comprises a different set of unique identifier molecules.Alternatively, or in addition, the disclosed methods may include a stepof incorporating a unique identifier molecule into each molecule withina particular discrete entity, e.g., droplet.

Printing Combined with Separation Techniques

In some embodiments, the printing methods described herein can becombined with separation techniques to achieve finer sensitivity fordetecting and analyzing the contents of a discrete entity. For example,discrete entities, e.g., droplets, containing cells can be printed to asubstrate and, for example, solidified by gelling them. The carrier oilcan then be removed and replaced with another material, such as a gelmatrix appropriate for separation. The substrate can then be incubatedunder conditions appropriate to lyse and separate the contents of thecells, such as by applying an electric current to induce electrophoreticmigration of molecules through the matrix in a specific direction, suchas perpendicular to the plane of the substrate or in the plane of thesubstrate. Such separation can also be performed in two or threedimensions, to achieve planar or cubic separations.

The spacing of the discrete entities, e.g., droplets, on the printedsubstrate can be selected so as to provide adequate distance for theseparation of the materials. The separated materials can then bedetected using a variety of techniques, such as blotting techniques suchas western, southern, and northern blotting, or optical orspectrographic techniques, such as Raman spectroscopy or massspectrometry, to name a few examples. This enables the contents of thecells to be analyzed with higher resolution by preforming a separationbased on biochemical properties prior to the analysis step, which is animportant aspect of sensitive measurement techniques like liquidchromatography mass spectrometry. The same concepts can apply to otherprinted entities, such as droplets encapsulating cell-free transcriptand translation reagents or other biological reagents, as well as toparticles and other liquid droplets in which such two-step separationand analysis techniques would be valuable. For example, such methods canbe used in synthetic biology screens by printing droplets encapsulatingcells or cell-free extracts expressing a target pathway and then usingthe separation procedure to separated important molecules in the mediumprior to performing the spectrographic or mass spectrometric analysis.

Accordingly, in some embodiments the present disclosure provides amethod, wherein the method includes printing discrete entities to asubstrate surface using any suitable method described herein; replacingthe carrier fluid with a material suitable for molecular separation,such as polyacrylamide, agarose, etc.; incubating the printed substrateunder conditions sufficient to induce molecular separation of thecontents of the discrete entities into the surrounding separationmaterial (e.g., by applying an electric current); and analyzing theseparated contents of the discrete entities in the material. In somesuch embodiments, the discrete entities include droplets includingbiological materials such as, for example, proteins, nucleic acids,cells, viruses, etc. In some embodiments, the separation is achievedusing gel electrophoresis. In some embodiments, the method may be usedto perform blotting experiments on the discrete entities and theircontents such as southern, northern, and western blotting. In someembodiments, the separated materials are analyzed using massspectrometry such as, for example, MALDI, NIMS, etc. In someembodiments, the materials are separated in more than one dimension bychanging the direction of the separating force. In some embodiments, theanalysis of the separated materials uses 3D imaging or other technique,such as confocal microscopy or layer-by-layer MALDI-MS to analyze theseparated materials in the 3D network of the separation material.

Devices

As indicated above, embodiments of the disclosed subject matter employsystems and/or devices including microfluidic devices. Devices of thesubject disclosure include all those described above in association withthe subject methods. Microfluidic devices of this disclosure may becharacterized in various ways.

In some aspects, for example, systems and/or devices are provided whichinclude one or more discrete entity, e.g., droplet, printers. Discreteentity printers may include one or more microfluidic device, such as amicrofluidic device including one or more discrete entity makers, e.g.,droplet makers, configured to generate discrete entities, e.g.,droplets, as described herein, and/or one or more flow channels. In someaspects, the one or more flow channels are operably connected, e.g.,fluidically connected, to the one or more droplet makers and/or areconfigured to receive one or more droplets therefrom. By “operablyconnected” and “operably coupled”, as used herein, is meant connected ina specific way (e.g., in a manner allowing fluid, e.g., water, to moveand/or electric power to be transmitted) that allows a disclosed systemor device and its various components to operate effectively in themanner described herein.

Aspects of the disclosed devices also include one or more deliveryorifice, such as a delivery orifice fluidically connected to one or moreflow channels. In some embodiments, delivery orifices include anopening, e.g., a circular or oblong opening, through which one or morediscrete entities may pass. In some embodiments, openings of deliveryorifices are defined by a rim of a device or a portion thereof, e.g., anozzle, such as a positionable nozzle. Delivery orifices, as included inthe subject embodiments, may have any of the same dimensions, e.g., across-sectional dimension, as the flow channels described herein, or mayhave different dimensions.

A delivery orifice as described herein, e.g., a delivery orifice of amicrofluidic nozzle as described herein, will generally have dimensionsthat are similar to the size of the droplets to be deliveredtherethrough. Accordingly, in some embodiments, a delivery orifice asdescribed herein has a diameter of from about 1 μm to about 1000 μm,inclusive, e.g., from about 10 μm to about 300 μm, inclusive. In someembodiments, a delivery orifice as described herein has a diameter offrom about 1 μm to about 10 μm, from about 10 μm to about 100 μm, fromabout 100 μm to about 500 μm, or from about 500 μm to about 1000 μm,inclusive.

The nozzle can be molded as part of a microfluidic sorter as describedherein, or can be a separate part that is mated with a microfluidicsorter as described herein. Suitable materials for the nozzle mayinclude, e.g., polymeric tubing, small bore hypodermic tubing, andmodified glass capillaries.

Embodiments of the subjects disclosure also include devices includingone or more automated system integrated with the delivery orifice,wherein the automated system (a) selectively positions, e.g., positionsby moving one or more distance on the order of magnitude of a discreteentity, the delivery orifice in proximity to a substrate or a portionthereof during operation and/or (b) selectively positions, e.g.,positions by moving one or more distance on the order of magnitude of adiscrete entity, the substrate or portion thereof in proximity to thedelivery orifice during operation, such that a discrete entity, e.g., adroplet, can be ejected from the delivery orifice and/or deposited onthe substrate. In some embodiments, automated systems are electronicand/or include one or more control unit for controlling automation, suchas a control unit including a central processing unit.

As noted above, droplet printers may include one or more flow channels,e.g., flow channels which discrete entities may pass into, out of,and/or through. In certain embodiments, flow channels are one or more“micro” channel. Such channels may have at least one cross-sectionaldimension on the order of a millimeter or smaller (e.g., less than orequal to about 1 millimeter). For certain applications, this dimensionmay be adjusted; in some embodiments the at least one cross-sectionaldimension is about 500 micrometers or less. In some embodiments, thecross-sectional dimension is about 100 micrometers or less, or about 10micrometers or less, and sometimes about 1 micrometer or less. Across-sectional dimension is one that is generally perpendicular to thedirection of centerline flow, although it should be understood that whenencountering flow through elbows or other features that tend to changeflow direction, the cross-sectional dimension in play need not bestrictly perpendicular to flow. It should also be understood that insome embodiments, a micro-channel may have two or more cross-sectionaldimensions such as the height and width of a rectangular cross-sectionor the major and minor axes of an elliptical cross-section. Either ofthese dimensions may be compared against sizes presented here. Note thatmicro-channels employed in this disclosure may have two dimensions thatare grossly disproportionate—e.g., a rectangular cross-section having aheight of about 100-200 micrometers and a width on the order or acentimeter or more. Of course, certain devices may employ channels inwhich the two or more axes are very similar or even identical in size(e.g., channels having a square or circular cross-section).

Microfluidic devices, in some embodiments of this disclosure, arefabricated using microfabrication technology. Such technology may beemployed to fabricate integrated circuits (ICs), microelectromechanicaldevices (MEMS), display devices, and the like. Among the types ofmicrofabrication processes that can be employed to produce smalldimension patterns in microfluidic device fabrication arephotolithography (including X-ray lithography, e-beam lithography,etc.), self-aligned deposition and etching technologies, anisotropicdeposition and etching processes, self-assembling mask formation (e.g.,forming layers of hydrophobic-hydrophilic copolymers), etc.

In view of the above, it should be understood that some of theprinciples and design features described herein can be scaled to largerdevices and systems including devices and systems employing channelsreaching the millimeter or even centimeter scale channel cross-sections.Thus, when describing some devices and systems as “microfluidic,” it isintended that the description apply equally, in certain embodiments, tosome larger scale devices.

When referring to a microfluidic “device” it is generally intended torepresent a single entity in which one or more channels, reservoirs,stations, etc. share a continuous substrate, which may or may not bemonolithic. Aspects of microfluidic devices include the presence of oneor more fluid flow paths, e.g., channels, having dimensions as discussedherein. A microfluidics “system” may include one or more microfluidicdevices and associated fluidic connections, electrical connections,control/logic features, etc.

For example, systems of the subject disclosure may include one or morediscrete entity printer, e.g., one or more droplet printer, and/or asubstrate or portion thereof, e.g., a substrate surface, for receivingone or more discrete entities, e.g., droplets deposited thereon by, forexample, a delivery orifice of a discrete entity printer, e.g., adroplet printer. Systems may also include one or more of: (a) atemperature control module for controlling the temperature of one ormore portions of the subject devices and/or discrete entities thereinand which is operably connected to the discrete entity printer, e.g., adroplet printer, (b) a detection means, i.e., a detector, e.g., anoptical imager, operably connected to the discrete entity printer, e.g.,a droplet printer, (c) an incubator, e.g., a cell incubator, operablyconnected to the discrete entity printer, e.g., a droplet printer, and(d) a sequencer operably connected to the discrete entity printer, e.g.,a droplet printer. The subject systems may also include one or moreconveyor configured to move, e.g., convey, a substrate from a firstdiscrete entity, e.g., droplet, receiving position to one or more of(a)-(d).

The subject devices and systems, include one or more sorter for sortingdiscrete entities, e.g., droplets, into one or more flow channels. Sucha sorter may sort and distribute discrete entities, e.g., droplets,based on one or more characteristics of the discrete entities includingcomposition, size, shape, buoyancy, or other characteristics.

Aspects of the devices also include one or more detection means i.e., adetector, e.g., an optical imager, configured for detecting the presenceof one or more discrete entities, e.g., droplets, or one or morecharacteristics thereof, including their composition. In someembodiments, detection means are configured to recognize one or morecomponents of one or more discrete entities, e.g., discrete entities, inone or more flow channel.

In various embodiments, microfluidic devices of this disclosure providea continuous flow of a fluid medium. Fluid flowing through a channel ina microfluidic device exhibits many unique properties. Typically, thedimensionless Reynolds number is extremely low, resulting in flow thatalways remains laminar. Further, in this regime, two fluids joining willnot easily mix, and diffusion alone may drive the mixing of twocompounds.

In addition, the subject devices, in some embodiments, include one ormore temperature and/or pressure control module. Such a module may becapable of modulating temperature and/or pressure of a carrier fluid inone or more flow channels of a device. More specifically, a temperaturecontrol module may be one or more thermal cycler.

Various features and examples of microfluidic device components suitablefor use with this disclosure will now be described.

Substrate

According to the subject disclosure, substrates used in microfluidicdevices and/or systems are the supports in which the necessary elementsfor fluid transport are provided. The basic structure of a substrate maybe monolithic, laminated, or otherwise sectioned. Substrates may includeone or more flow channels, such as microchannels serving as conduits formolecular libraries and/or reagents. They may also include input ports,output ports, and/or features to assist in flow control.

In certain embodiments, the substrate choice may be dependent on theapplication and design of the device. Substrate materials may be chosenfor their compatibility with a variety of operating conditions.Limitations in microfabrication processes for a given material are alsorelevant considerations in choosing a suitable substrate. Usefulsubstrate materials which may be employed with the subject disclosureinclude, e.g., glass, polymers, silicon, metal, ceramics, and/orcombinations thereof.

The subject devices, in some embodiments, include one or more polymers.Polymers are useful materials for microfluidic devices because they areamenable to both cost effective and high volume production. Polymers,including polymers for use in accordance with the subject disclosure,can be classified into three categories according to their moldingbehavior: thermoplastic polymers, elastomeric polymers and duroplasticpolymers. Thermoplastic polymers can be molded into shapes above theglass transition temperature, and will retain these shapes after coolingbelow the glass transition temperature. Elastomeric polymers can bestretched upon application of an external force, but will go back tooriginal state once the external force is removed. Elastomers do notmelt before reaching their decomposition temperatures. Duroplasticpolymers have to be cast into their final shape because they soften alittle before the temperature reaches their decomposition temperature.

Among the polymers that may be used in microfabricated device of thisdisclosure are polyamide (PA), polybutylenterephthalate (PBT),polycarbonate (PC), polyethylene (PE), polymethylmethacrylate (PMMA),polyoxymethylene (POM), polypropylene (PP), polyphenylenether (PPE),polystyrene (PS) and polysulphone (PSU). The chemical and physicalproperties of polymers can limit their uses in microfluidic devices.Specifically in comparison to glass, the lower resistance againstchemicals, the aging, the mechanical stability, and the UV stability canlimit the use of polymers for certain applications.

Glass, which may also be used as the substrate material, has specificadvantages under certain operating conditions. Since glass is chemicallyinert to most liquids and gases, it is particularly appropriate forapplications employing certain solvents that have a tendency to dissolveplastics. Additionally, its transparent properties make glassparticularly useful for optical or UV detection.

Surface Treatments and Coatings

Surface modification may be useful for controlling the functionalmechanics (e.g., flow control) of a microfluidic device and may beapplied according to the subject disclosure. For example, it may beuseful to keep fluidic species from adsorbing to channel walls or forattaching antibodies to the surface for detection of biologicalcomponents.

Polymer devices in particular tend to be hydrophobic, and thus loadingof the channels may be difficult. The hydrophobic nature of polymersurfaces may also make it difficult to control electroosmotic flow(EOF). One technique for coating polymer surface according to thesubject disclosure is the application of polyelectrolyte multilayers(PEM) to channel surfaces. PEM involves filling the channel successivelywith alternating solutions of positive and negative polyelectrolytesallowing for multilayers to form electrostatic bonds. Although thelayers typically do not bond to the channel surfaces, they maycompletely cover the channels even after long-term storage. Anothertechnique for applying a hydrophilic layer on polymer surfaces accordingto the subject disclosure involves the UV grafting of polymers to thesurface of the channels. First grafting sites, radicals, are created atthe surface by exposing the surface to UV irradiation whilesimultaneously exposing the device to a monomer solution. The monomersreact to form a polymer covalently bonded at the reaction site.

In some embodiments, glass channels according to the subject disclosure,generally have high levels of surface charge, thereby causing proteinsto adsorb and possibly hindering separation processes. In somesituations, the disclosure includes applying a polydimethylsiloxane(PDMS) and/or surfactant coating to the glass channels. Other polymersthat may be employed to retard surface adsorption includepolyacrylamide, glycol groups, polysiloxanes, glyceroglycidoxypropyl,poly(ethyleneglycol) and hydroxyethylated poly(ethyleneimine).Furthermore, subject electroosmotic devices may include a coatingbearing a charge that is adjustable in magnitude by manipulatingconditions inside of the device (e.g. pH). The direction of the flow canalso be selected based on the coating since the coating can either bepositively or negatively charged.

Specialized coatings can also be applied according to this disclosure toimmobilize certain species on the channel surface—this process is called“functionalizing the surface.” For example, a polymethylmethacrylate(PMMA) surface may be coated with amines to facilitate attachment of avariety of functional groups or targets. Alternatively, PMMA surfacescan be rendered hydrophilic through an oxygen plasma treatment process.

Microfluidic Elements

Microfluidic systems and devices according to the subject disclosure cancontain one or more flow channels, such as microchannels, valves, pumps,reactors, mixers and other/or components. Some of these components andtheir general structures and dimensions are discussed below.

Various types of valves can be applied for flow control in microfluidicdevices of this disclosure. These include, but are not limited topassive valves and check valves (membrane, flap, bivalvular, leakage,etc.). Flow rate through these valves are dependent on various physicalfeatures of the valve such as surface area, size of flow channel, valvematerial, etc. Valves also have associated operational and manufacturingadvantages/disadvantages that may be taken into consideration duringdesign of a microfluidic device.

Embodiments of the subject devices include one or more micropumps.Micropumps, as with other microfluidic components, are subjected tomanufacturing constraints. Typical considerations in pump design includetreatment of bubbles, clogs, and durability. Micropumps which may beincluded in the subject devices include, but are not limited to electricequivalent pumps, fixed-stroke microdisplacement, peristalticmicromembrane and/or pumps with integrated check valves.

Macrodevices rely on turbulent forces such as shaking and stirring tomix reagents. In comparison, such turbulent forces are not practicallyattainable in microdevices, such as those of the present disclosure, andinstead mixing in microfluidic devices is generally accomplished throughdiffusion. Since mixing through diffusion can be slow and inefficient,microstructures, such as those employed with the disclosed subjectmatter, are often designed to enhance the mixing process. Thesestructures manipulate fluids in a way that increases interfacial surfacearea between the fluid regions, thereby speeding up diffusion. Incertain embodiments, microfluidic mixers are employed. Such mixers maybe provided upstream from, and in some cases integrated with, amicrofluidic separation device and/or a sorter, of this disclosure.

In some embodiments, the devices and systems of the present disclosureinclude micromixers. Micromixers may be classified into two generalcategories: active mixers and passive mixers. Active mixers work byexerting active control over flow regions (e.g. varying pressuregradients, electric charges, etc.). Passive mixers do not requireinputted energy and use only “fluid dynamics” (e.g. pressure) to drivefluid flow at a constant rate. One example of a passive mixer involvesstacking two flow streams on top of one another separated by a plate.The flow streams are contacted with each other once the separation plateis removed. The stacking of the two liquids increases contact area anddecreases diffusion length, thereby enhancing the diffusion process.Mixing and reaction devices can be connected to heat transfer systems ifheat management is needed. As with macro-heat exchangers, micro-heatexchanges can either have co-current, counter-current, or cross-flowflow schemes. Microfluidic devices may have channel widths and depthsbetween about 10 μm and about 10 cm. One channel structure includes along main separation channel, and three shorter “offshoot” side channelsterminating in either a buffer, sample, or waste reservoir. Theseparation channel can be several centimeters long, and the three sidechannels usually are only a few millimeters in length. Of course, theactual length, cross-sectional area, shape, and branch design of amicrofluidic device depends on the application as well other designconsiderations such as throughput (which depends on flow resistance),velocity profile, residence time, etc.

Microfluidic devices described herein may include one or more electricfield generators to perform certain steps of the methods describedherein, including, but not limited to, picoinjection, dropletcoalescence, selective droplet fusion, and droplet sorting. In certainembodiments, the electric fields are generated using metal electrodes.In particular embodiments, electric fields are generated using liquidelectrodes. In certain embodiments, liquid electrodes include liquidelectrode channels filled with a conducting liquid (e.g. salt water orbuffer) and situated at positions in the microfluidic device where anelectric field is desired. In particular embodiments, the liquidelectrodes are energized using a power supply or high voltage amplifier.In some embodiments, the liquid electrode channel includes an inlet portso that a conducting liquid can be added to the liquid electrodechannel. Such conducting liquid may be added to the liquid electrodechannel, for example, by connecting a tube filled with the liquid to theinlet port and applying pressure. In particular embodiments, the liquidelectrode channel also includes an outlet port for releasing conductingliquid from the channel. In particular embodiments, the liquidelectrodes are used in picoinjection, droplet coalescence, selectivedroplet fusion, and/or droplet sorting aspects of a microfluidic devicedescribed herein. Liquid electrodes may find use, for example, where amaterial to be injected via application of an electric field is notcharged.

In certain embodiments, the width of one or more of the microchannels ofthe microfluidic device (e.g., input microchannel, pairing microchannel,pioinjection microchannel, and/or a flow channel upstream or downstreamof one or more of these channels) is 100 microns or less, e.g, 90microns or less, 80 microns or less, 70 microns or less, 60 microns orless, 50 microns or less, e.g., 45 microns or less, 40 microns or less,39 microns or less, 38 microns or less, 37 microns or less, 36 micronsor less, 35 microns or less, 34 microns or less, 33 microns or less, 32microns or less, 31 microns or less, 30 microns or less, 29 microns orless, 28 microns or less, 27 microns or less, 26 microns or less, 25microns or less, 20 microns or less, 15 microns or less, or 10 micronsor less. In some embodiments, the width of one or more of the abovemicrochannels is from about 10 microns to about 15 microns, from about15 microns to about 20 microns, from about 20 microns to about 25microns, from about 25 microns to about 30 microns, from about 30microns to about 35 microns, from about 35 microns to about 40 microns,from about 40 microns to about 45 microns, or from about 45 microns toabout 50 microns, from about 50 microns to about 60 microns, from about60 microns to about 70 microns, from about 70 microns to about 80microns, from about 80 microns to about 90 microns, or from about 90microns to about 100 microns.

-   Additional descriptions of various microchannel structures and    features which may be utilized in connection with the disclosed    methods and devices are provided in PCT Publication No. WO    2014/028378, the disclosure of which is incorporated by reference    herein in its entirety and for all purposes.

Methods of Fabrication

According to the disclosed embodiments, microfabrication processesdiffer depending on the type of materials used in the substrate and/orthe desired production volume. For small volume production orprototypes, fabrication techniques include LIGA, powder blasting, laserablation, mechanical machining, electrical discharge machining,photoforming, etc. Technologies for mass production of microfluidicdevices may use either lithographic or master-based replicationprocesses. Lithographic processes for fabricating substrates fromsilicon/glass include both wet and dry etching techniques commonly usedin fabrication of semiconductor devices. Injection molding and hotembossing typically are used for mass production of plastic substrates.

Glass, Silicon and Other “Hard” Materials (Lithography, Etching,Deposition)

According to embodiments of the disclosed subject matter, a combinationof lithography, etching and/or deposition techniques may be used to makemicrocanals and microcavities out of glass, silicon and other “hard”materials. Technologies based on the above techniques may be applied infabrication of devices in the scale of 0.1-500 micrometers.

Microfabrication techniques based on semiconductor fabrication processesare generally carried out in a clean room. The quality of the clean roomis classified by the number of particles <4 μm in size in a cubic inch.Typical clean room classes for MEMS microfabrication may be 1000 to10000.

In certain embodiments, photolithography may be used inmicrofabrication. In photolithography, a photoresist that has beendeposited on a substrate is exposed to a light source through an opticalmask. Conventional photoresist methods allow structural heights of up to10-40 μm. If higher structures are needed, thicker photoresists such asSU-8, or polyimide, which results in heights of up to 1 mm, can be used.

After transferring the pattern on the mask to the photoresist-coveredsubstrate, the substrate is then etched using either a wet or dryprocess. In wet etching, the substrate—area not protected by the mask—issubjected to chemical attack in the liquid phase. The liquid reagentused in the etching process depends on whether the etching is isotropicor anisotropic. Isotropic etching generally uses an acid to formthree-dimensional structures such as spherical cavities in glass orsilicon. Anisotropic etching forms flat surfaces such as wells andcanals using a highly basic solvent. Wet anisotropic etching on siliconcreates an oblique channel profile.

Dry etching involves attacking the substrate by ions in either a gaseousor plasma phase. Dry etching techniques can be used to createrectangular channel cross-sections and arbitrary channel pathways.Various types of dry etching that may be employed including physical,chemical, physico-chemical (e.g., RIE), and physico-chemical withinhibitor. Physical etching uses ions accelerated through an electricfield to bombard the substrate's surface to “etch” the structures.Chemical etching may employ an electric field to migrate chemicalspecies to the substrate's surface. The chemical species then reactswith the substrate's surface to produce voids and a volatile species.

In certain embodiments, deposition is used in microfabrication.Deposition techniques can be used to create layers of metals,insulators, semiconductors, polymers, proteins and other organicsubstances. Most deposition techniques fall into one of two maincategories: physical vapor deposition (PVD) and chemical vapordeposition (CVD). In one approach to PVD, a substrate target iscontacted with a holding gas (which may be produced by evaporation forexample). Certain species in the gas adsorb to the target's surface,forming a layer constituting the deposit. In another approach commonlyused in the microelectronics fabrication industry, a target containingthe material to be deposited is sputtered with using an argon ion beamor other appropriately energetic source. The sputtered material thendeposits on the surface of the microfluidic device. In CVD, species incontact with the target react with the surface, forming components thatare chemically bonded to the object. Other deposition techniquesinclude: spin coating, plasma spraying, plasma polymerization, dipcoating, casting and Langmuir-Blodgett film deposition. In plasmaspraying, a fine powder containing particles of up to 100 μm in diameteris suspended in a carrier gas. The mixture containing the particles isaccelerated through a plasma jet and heated. Molten particles splatteronto a substrate and freeze to form a dense coating. Plasmapolymerization produces polymer films (e.g. PMMA) from plasma containingorganic vapors.

Once the microchannels, microcavities and other features have beenetched into the glass or silicon substrate, the etched features areusually sealed to ensure that the microfluidic device is “watertight.”When sealing, adhesion can be applied on all surfaces brought intocontact with one another. The sealing process may involve fusiontechniques such as those developed for bonding between glass-silicon,glass-glass, or silicon-silicon.

Anodic bonding can be used for bonding glass to silicon. A voltage isapplied between the glass and silicon and the temperature of the systemis elevated to induce the sealing of the surfaces. The electric fieldand elevated temperature induces the migration of sodium ions in theglass to the glass-silicon interface. The sodium ions in theglass-silicon interface are highly reactive with the silicon surfaceforming a solid chemical bond between the surfaces. The type of glassused may have a thermal expansion coefficient near that of silicon (e.g.Pyrex Corning 7740).

Fusion bonding can be used for glass-glass or silicon-silicon sealing.The substrates are first forced and aligned together by applying a highcontact force. Once in contact, atomic attraction forces (primarily vander Waals forces) hold the substrates together so they can be placedinto a furnace and annealed at high temperatures. Depending on thematerial, temperatures used ranges between about 600 and 1100° C.

Polymers/Plastics

A variety of techniques may be employed for micromachining plasticsubstrates in accordance with the subject embodiments. Among these arelaser ablation, stereolithography, oxygen plasma etching, particle jetablation, and microelectro-erosion. Some of these techniques can be usedto shape other materials (glass, silicon, ceramics, etc.) as well.

To produce multiple copies of a microfluidic device, replicationtechniques are employed. Such techniques involve first fabricating amaster or mold insert containing the pattern to be replicated. Themaster is then used to mass-produce polymer substrates through polymerreplication processes.

In the replication process, the master pattern contained in a mold isreplicated onto the polymer structure. In certain embodiments, a polymerand curing agent mix is poured onto a mold under high temperatures.After cooling the mix, the polymer contains the pattern of the mold, andis then removed from the mold. Alternatively, the plastic can beinjected into a structure containing a mold insert. In microinjection,plastic heated to a liquid state is injected into a mold. Afterseparation and cooling, the plastic retains the mold's shape.

PDMS (polydimethylsiloxane), a silicon-based organic polymer, may beemployed in the molding process to form microfluidic structures. Becauseof its elastic character, PDMS is suited for microchannels between about5 μm and 500 μm. Specific properties of PDMS make it suitable formicrofluidic purposes. Such properties include:

-   -   1) It is optically clear which allows for visualization of the        flows.    -   2) PDMS, when mixed with a proper amount of reticulating agent,        has elastomeric qualities that facilitates keeping microfluidic        connections “watertight.”    -   3) Valves and pumps using membranes can be made with PDMS        because of its elasticity.    -   4) Untreated PDMS is hydrophobic, and becomes temporarily        hydrophilic after oxidation of surface by oxygen plasma or after        immersion in strong base; oxidized PDMS adheres by itself to        glass, silicon, or polyethylene, as long as those surfaces were        themselves exposed to an oxygen plasma.    -   5) PDMS is permeable to gas. Filling of the channel with liquids        is facilitated even when there are air bubbles in the canal        because the air bubbles are forced out of the material.        Additionally, PDMS is also permeable to non polar-organic        solvents.

Microinjection can be used to form plastic substrates employed in a widerange of microfluidic designs. In this process, a liquid plasticmaterial is first injected into a mold under vacuum and pressure, at atemperature greater than the glass transition temperature of theplastic. The plastic is then cooled below the glass transitiontemperature. After removing the mold, the resulting plastic structure isthe negative of the mold's pattern.

Yet another replicating technique is hot embossing, in which a polymersubstrate and a master are heated above the polymer's glass transitiontemperature, Tg (which for PMMA or PC is around 100-180° C.). Theembossing master is then pressed against the substrate with a presetcompression force. The system is then cooled below Tg and the mold andsubstrate are then separated.

Typically, the polymer is subjected to the highest physical forces uponseparation from the mold tool, particularly when the microstructurecontains high aspect ratios and vertical walls. To avoid damage to thepolymer microstructure, material properties of the substrate and themold tool may be taken into consideration. These properties include:sidewall roughness, sidewall angles, chemical interface betweenembossing master and substrate and temperature coefficients. Highsidewall roughness of the embossing tool can damage the polymermicrostructure since roughness contributes to frictional forces betweenthe tool and the structure during the separation process. Themicrostructure may be destroyed if frictional forces are larger than thelocal tensile strength of the polymer. Friction between the tool and thesubstrate may be important in microstructures with vertical walls. Thechemical interface between the master and substrate could also be ofconcern. Because the embossing process subjects the system to elevatedtemperatures, chemical bonds could form in the master-substrateinterface. These interfacial bonds could interfere with the separationprocess. Differences in the thermal expansion coefficients of the tooland the substrate could create addition frictional forces.

Various techniques can be employed to form molds, embossing masters, andother masters containing patterns used to replicate plastic structuresthrough the replication processes mentioned above. Examples of suchtechniques include LIGA (described below), ablation techniques, andvarious other mechanical machining techniques. Similar techniques canalso be used for creating masks, prototypes and microfluidic structuresin small volumes. Materials used for the mold tool include metals, metalalloys, silicon and other hard materials.

Laser ablation may be employed to form microstructures either directlyon the substrate or through the use of a mask. This technique uses aprecision-guided laser, typically with wavelength between infrared andultraviolet. Laser ablation may be performed on glass and metalsubstrates, as well as on polymer substrates. Laser ablation can beperformed either through moving the substrate surface relative to afixed laser beam, or moving the beam relative to a fixed substrate.Various micro-wells, canals, and high aspect structures can be made withlaser ablation.

Certain materials, such as stainless steel, make durable mold insertsand can be micromachined to form structures down to the 10-μm range.Various other micromachining techniques for microfabrication existincluding μ-Electro Discharge Machining (μ-EDM), μ-milling, focused ionbeam milling. μ-EDM allows the fabrication of 3-dimensional structuresin conducting materials. In μ-EDM, material is removed by high-frequencyelectric discharge generated between an electrode (cathode tool) and aworkpiece (anode). Both the workpiece and the tool are submerged in adielectric fluid. This technique produces a comparatively roughersurface but offers flexibility in terms of materials and geometries.

Electroplating may be employed for making a replication mold tool/masterout of, e.g., a nickel alloy. The process starts with a photolithographystep where a photoresist is used to defined structures forelectroplating. Areas to be electroplated are free of resist. Forstructures with high aspect ratios and low roughness requirements, LIGAcan be used to produce electroplating forms. LIGA is a German acronymfor Lithographic (Lithography), Galvanoformung (electroplating),Abformung (molding). In one approach to LIGA, thick PMMA layers areexposed to x-rays from a synchrotron source. Surfaces created by LIGAhave low roughness (around 10 nm RMS) and the resulting nickel tool hasgood surface chemistry for most polymers.

As with glass and silicon devices, polymeric microfluidic devices mustbe closed up before they can become functional. Common problems in thebonding process for microfluidic devices include the blocking ofchannels and changes in the physical parameters of the channels.Lamination is one method used to seal plastic microfluidic devices. Inone lamination process, a PET foil (about 30 μm) coated with a meltingadhesive layer (typically 5 μm-10 μm) is rolled with a heated roller,onto the microstructure. Through this process, the lid foil is sealedonto the channel plate. Several research groups have reported a bondingby polymerization at interfaces, whereby the structures are heated andforce is applied on opposite sides to close the channel. But excessiveforce applied may damage the microstructures. Both reversible andirreversible bonding techniques exist for plastic-plastic andplastic-glass interfaces. One method of reversible sealing involvesfirst thoroughly rinsing a PDMS substrate and a glass plate (or a secondpiece of PDMS) with methanol and bringing the surfaces into contact withone another prior to drying. The microstructure is then dried in an ovenat 65° C. for 10 min. No clean room is required for this process.Irreversible sealing is accomplished by first thoroughly rinsing thepieces with methanol and then drying them separately with a nitrogenstream. The two pieces are then placed in an air plasma cleaner andoxidized at high power for about 45 seconds. The substrates are thenbrought into contact with each other and an irreversible seal formsspontaneously.

Other available techniques include laser and ultrasonic welding. Inlaser welding, polymers are joined together through laser-generatedheat. This method has been used in the fabrication of micropumps.Ultrasonic welding is another bonding technique that may be employed insome applications.

One nucleic acid amplification technique described herein is apolymerase chain reaction (PCR). However, in certain embodiments,non-PCR amplification techniques may be employed such as variousisothermal nucleic acid amplification techniques; e.g., real-time stranddisplacement amplification (SDA), rolling-circle amplification (RCA) andmultiple-displacement amplification (MDA).

Regarding PCR amplification modules, it will be necessary to provide tosuch modules at least the building blocks for amplifying nucleic acids(e.g., ample concentrations of four nucleotides), primers, polymerase(e.g., Taq), and appropriate temperature control programs). Thepolymerase and nucleotide building blocks may be provided in a buffersolution provided via an external port to the amplification module orfrom an upstream source. In certain embodiments, the buffer streamprovided to the sorting module contains some of all the raw materialsfor nucleic acid amplification. For PCR in particular, precisetemperature control of the reacting mixture is extremely important inorder to achieve high reaction efficiency. One method of on-chip thermalcontrol is Joule heating in which electrodes are used to heat the fluidinside the module at defined locations. The fluid conductivity may beused as a temperature feedback for power control.

In certain aspects, the discrete entities, e.g., microdroplets,containing the PCR mix may be flowed through a channel that incubatesthe discrete entities under conditions effective for PCR. Flowing thediscrete entities through a channel may involve a channel that snakesover various temperature zones maintained at temperatures effective forPCR. Such channels may, for example, cycle over two or more temperaturezones, wherein at least one zone is maintained at about 65° C. and atleast one zone is maintained at about 95° C. As the discrete entitiesmove through such zones, their temperature cycles, as needed for PCR.The precise number of zones, and the respective temperature of eachzone, may be readily determined by those of skill in the art to achievethe desired PCR amplification.

Exemplary Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-252 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below.

-   -   1. A method of delivering discrete entities to a substrate, the        method including:        -   flowing a plurality of discrete entities through a            microfluidic device in a carrier fluid, wherein the discrete            entities are insoluble and/or immiscible in the carrier            fluid;        -   directing the carrier fluid and one or more of the plurality            of discrete entities through a delivery orifice to the            substrate; and        -   affixing the one or more of the plurality of discrete            entities to the substrate.    -   2. The method of 1, wherein the one or more of the plurality of        discrete entities are affixed to the substrate via a force,        wherein the force is selected from a gravitational force, an        electrical force, a magnetic force, and combinations thereof.    -   3. The method of 2, including storing the affixed entity under        controlled environmental conditions for a storage period,        wherein the force is maintained during the storage period.    -   4. The method of 3, wherein the controlled environmental        conditions include a constant temperature and/or pressure.    -   5. The method of any one of 2-4, wherein the force is an        electrical force.    -   6. The method of 5, wherein the electrical force is a        dielectrophoretic force.    -   7. The method of any one of 1-6, wherein the discrete entities        are droplets.    -   8. The method of 7, wherein the droplets are affixed to the        substrate via wetting.    -   9. The method of 7, wherein the droplets include an aqueous        fluid, which is immiscible with the carrier fluid.    -   10. The method of 9, wherein the substrate includes on a first        surface a layer of fluid which is miscible with the carrier        fluid and immiscible with the aqueous fluid, and wherein the        droplets are affixed to the first surface of the substrate        following introduction into the layer of fluid on the first        surface of the substrate.    -   11. The method of 7, wherein the carrier fluid is an aqueous        fluid and the droplets include a fluid which is immiscible with        the carrier fluid.    -   12. The method of 11, wherein the substrate includes on a first        surface a layer of aqueous fluid which is miscible with the        carrier fluid and immiscible with the fluid included by the        droplets, and wherein the droplets are affixed to the first        surface of the substrate following introduction into the layer        of aqueous fluid on the first surface of the substrate.    -   13. The method of 1, wherein the discrete entities are affixed        to the substrate via interfacial tension.    -   14. The method of any one of 1-13, wherein the discrete entities        have a dimension of from about 1 to 1000 μm.    -   15. The method of 14, wherein the discrete entities have a        diameter of from about 1 to 1000 μm.    -   16. The method of any one of 1-13, wherein the discrete entities        have a volume of from about 1 femtoliter to about 1000        nanoliters.    -   17. The method of any one of 1-16, wherein the microfluidic        device includes a sorter, and wherein the method includes        sorting, via the sorter, the one or more of the plurality of        discrete entities to be delivered through the delivery orifice        to the substrate from the plurality of discrete entities.    -   18. The method of 17, wherein the sorter includes a flow channel        including a gapped divider including a separating wall which        extends less than the complete height of the flow channel.    -   19. The method of 17, wherein the plurality of discrete entities        is optically scanned prior to the sorting.    -   20. The method of 19, wherein the sorter includes an optical        fiber configured to apply excitation energy to one or more of        the plurality of discrete entities.    -   21. The method of 20, wherein the sorter includes a second        optical fiber configured to collect a signal produced by the        application of excitation energy to one or more of the plurality        of discrete entities.    -   22. The method of 20, wherein the optical fiber is configured to        apply excitation energy to one or more of the plurality of        discrete entities and collect a signal produced by the        application of the excitation energy to one or more of the        plurality of discrete entities.    -   23. The method of 19, wherein the sorting is based on results        obtained from the optical scan.    -   24. The method of 17, wherein the sorter is an active sorter.    -   25. The method of 17, wherein the sorter is a passive sorter.    -   26. The method of 24, wherein the sorting includes sorting via        dielectrophoresis.    -   27. The method of 24, wherein the sorter includes one or more        microfluidic valves, and wherein the sorting includes sorting        via activation of the one or more microfluidic valves.    -   28. The method of any one of 1-27, wherein the discrete entities        are droplets, the microfluidic device includes a selectively        activatable droplet maker which forms droplets from a fluid        stream, and wherein the method includes forming one or more of        the plurality of discrete entities via selective activation of        the droplet maker.    -   29. The method of any one of 1-28, wherein the plurality of        discrete entities includes discrete entities which differ in        composition.    -   30. The method of any one of 1-29, wherein the microfluidic        device is integrated with an automated system which selectively        positions the delivery orifice relative to the substrate, and        wherein the method includes selectively positioning via the        automated system the delivery orifice relative to the substrate        to selectively deliver the one or more of the plurality of        discrete entities to one or more locations on or in proximity to        the substrate.    -   31. The method of any one of 1-29, wherein the microfluidic        device is integrated with an automated system which selectively        positions the substrate relative to the delivery orifice, and        wherein the method includes selectively positioning via the        automated system the substrate relative to the delivery orifice        to selectively deliver the one or more of the plurality of        discrete entities to one or more locations on or in proximity to        the substrate.    -   32. The method of 30 or 31, wherein the method includes        delivering a first member of the plurality of discrete entities        to a first location on or in proximity to the substrate and a        second member of the plurality of discrete entities to a second        location on or in proximity to the substrate.    -   33. The method of 32, wherein the first and second locations are        the same.    -   34. The method of any one of 1-33, wherein one or more        biological assays are performed in one or more of the discrete        entities before and/or after delivery to the substrate.    -   35. The method of any one of 1-34, wherein the temperature of        one or more of the discrete entities is controlled before and/or        after delivery to the substrate.    -   36. The method of 35, wherein one or more of the discrete        entities are thermalcycled before and/or after delivery to the        substrate.    -   37. The method of any one of 17-36, wherein the members of the        plurality of discrete entities which are not sorted for delivery        through the delivery orifice to the substrate are recovered.    -   38. The method of 37, wherein the recovered members of the        plurality of discrete entities are recycled such that the method        of 1 is repeated with the recovered members of the plurality of        discreet entities.    -   39. The method of 38, wherein recovered members of the plurality        of discrete entities are continuously recycled during        performance of the method.    -   40. The method of any one of 1-39, wherein one or more of the        plurality of discrete entities includes a cell.    -   41. The method 40, wherein each member of the plurality of        discrete entities includes not more than one cell.    -   42. The method of any one of 1-39, wherein one or more of the        plurality of discrete entities includes a nucleic acid.    -   43. The method of any one of 1-42, wherein the method includes        encapsulating or incorporating one or more reagents into the        plurality of discrete entities.    -   44. The method of 43, wherein the one or more reagents include        amplification reagents.    -   45. The method of 44, wherein the amplification reagents include        Polymerase Chain Reaction (PCR) reagents.    -   46. A method of printing one or more cell layers, the method        including:        -   encapsulating cells in droplets including an aqueous fluid            to provide cell-including droplets;        -   flowing a plurality of droplets including the cell-including            droplets through a microfluidic device in a carrier fluid,            wherein the carrier fluid is immiscible with the aqueous            fluid;        -   directing the carrier fluid and a plurality of the            cell-including droplets through a delivery orifice to a            substrate; and        -   affixing the plurality of the cell-including droplets to the            substrate to provide a first layer of cell-including            droplets, wherein the substrate includes on a first surface            a layer of fluid which is miscible with the carrier fluid            and immiscible with the aqueous fluid, and wherein the            plurality of the cell-including droplets are affixed to the            first surface of the substrate following introduction into            the layer of fluid on the first surface of the substrate.    -   47. The method of 46, wherein the microfluidic device includes a        sorter, and wherein the method includes sorting, via the sorter,        the plurality of cell-including droplets to be delivered through        the delivery orifice to the substrate from the cell-including        droplets.    -   48. The method of 47, wherein the sorter includes a flow channel        including a gapped divider including a separating wall which        extends less than the complete height of the flow channel.    -   49. The method of any one of 46-48, wherein the method includes        -   encapsulating or incorporating one or more reagents into            droplets to provide reagent-including droplets;        -   flowing a plurality of droplets including the            reagent-including droplets through the microfluidic device            in the carrier fluid;        -   and directing the carrier fluid and a plurality of the            reagent-including droplets through the delivery orifice to            the substrate.    -   50. The method of 49, wherein the reagent-including droplets and        the cell-including droplets are the same.    -   51. The method of 49, wherein cell-including droplets and        reagent-including droplets are deposited in the same layer on        the substrate.    -   52. The method of any one of 49-51, wherein the one or more        reagents include a material which facilitates cell growth.    -   53. The method of 52, wherein the material which facilitates        cell growth includes a cell culture media component.    -   54. The method of 52, wherein the material which facilitates        cell growth includes a cell culture substrate.    -   55. The method of any one of 46-54, wherein the method includes        -   directing the carrier fluid and a second plurality of the            cell-including droplets through the delivery orifice to the            substrate; and        -   depositing a second layer of cell-including droplets on the            first layer of cell-including droplets thereby providing a            layered structure.    -   56. The method of any one of 46-54, wherein the method includes        -   depositing a layer of reagent-including droplets on the            first layer of cell-including droplets thereby providing a            layered structure.    -   57. A method of printing and detecting one or more cells, the        method including:        -   encapsulating cells in droplets including an aqueous fluid            to provide cell-including droplets;        -   flowing a plurality of droplets including the cell-including            droplets through a microfluidic device in a carrier fluid,            wherein the carrier fluid is immiscible with the aqueous            fluid;        -   directing the carrier fluid and a plurality of the            cell-including droplets through a delivery orifice to a            substrate;        -   affixing the plurality of the cell-including droplets to the            substrate, wherein the substrate includes on a first surface            a layer of fluid which is miscible with the carrier fluid            and immiscible with the aqueous fluid, and wherein the            plurality of the cell-including droplets are affixed to the            first surface of the substrate following introduction into            the layer of fluid on the first surface of the substrate;            and        -   detecting one or more of the cells in the affixed            cell-including droplets, a component of one or more of the            cells in the affixed cell-including droplets, or a product            of one or more of the cells in the affixed cell-including            droplets.    -   58. The method of 57, wherein the detecting is performed at a        plurality of time points.    -   59. The method of 57, wherein the method includes continuously        detecting over a period of time one or more of the cells in the        affixed cell-including droplets, a component of one or more of        the cells in the affixed cell-including droplets, or a product        of one or more of the cells in the affixed cell-including        droplets.    -   60. The method of any one of 57-59, including recovering from        the affixed cell-including droplets one or more of the cells in        the affixed cell-including droplets, a component of one or more        of the cells in the affixed cell-including droplets, or a        product of one or more of the cells in the affixed        cell-including droplets.    -   61. The method of 60, including recovering DNA from one or more        of the cells in the affixed cell-including droplets.    -   62. The method of 61, including sequencing the DNA.    -   63. A method of printing a three-dimensional structure, the        method including:        -   flowing discrete entities through a microfluidic device in a            carrier fluid, wherein the discrete entities are insoluble            and/or immiscible in the carrier fluid; and        -   directing the carrier fluid and a first plurality of the            discrete entities through a delivery orifice to a substrate            to provide a first layer thereon;        -   directing the carrier fluid and a second plurality of the            discrete entities through the delivery orifice to the first            layer to provide a second layer thereon; and        -   one or more additional directing steps in which a plurality            of the discrete entities are directed through the delivery            orifice to an immediately preceding layer to provide a            subsequent layer thereon, wherein a multilayer,            three-dimensional structure is provided.    -   64. The method of 63, wherein the discrete entities are        hydrophilic and the carrier fluid is hydrophobic.    -   65. The method of 64, wherein the discrete entities are        droplets.    -   66. The method of 65, wherein the droplets include an aqueous        fluid.    -   67. The method of 66, wherein the substrate includes on a first        surface a layer of fluid which is miscible with the carrier        fluid and immiscible with the aqueous fluid, and wherein the        droplets are affixed to the first surface of the substrate        following introduction into the layer of fluid on the first        surface of the substrate.    -   68. The method of 63, wherein the discrete entities are        hydrophobic and the carrier fluid is hydrophilic.    -   69. The method of 68, wherein the discrete entities are        droplets.    -   70. The method of 69, wherein the carrier fluid is an aqueous        fluid and the droplets include a fluid which is immiscible with        the carrier fluid.    -   71. The method of 70, wherein the substrate includes on a first        surface a layer of aqueous fluid which is miscible with the        carrier fluid and immiscible with the fluid included by the        droplets, and wherein the droplets are affixed to the first        surface of the substrate following introduction into the layer        of aqueous fluid on the first surface of the substrate.    -   72. The method of 64 or 68, wherein the discrete entities        consist of a solid material.    -   73. The method of 64 or 68, wherein the discrete entities        consist of a gel material.    -   74. The method of 64 or 68, including initiating a reaction        which causes the discrete entities or the carrier fluid to        solidify.    -   75. The method of 74, wherein the reaction is a        photopolymerization reaction.    -   76. A method of delivering droplets from a delivery orifice, the        method including:        -   flowing a plurality of droplets through a microfluidic            device in a carrier fluid, wherein the microfluidic device            includes a sorter;        -   detecting one or more of the plurality of droplets to            provide one or more detected droplets;        -   sorting via the sorter the one or more detected droplets            from the plurality of droplets;        -   directing the carrier fluid and the one or more detected            droplets through the delivery orifice.    -   77. The method of 76, including depositing the one or more        detected droplets on a substrate.    -   78. The method of 76 or 77, wherein the droplets include an        aqueous fluid, which is immiscible with the carrier fluid.    -   79. The method of 78, wherein the substrate includes on a first        surface a layer of fluid which is miscible with the carrier        fluid and immiscible with the aqueous fluid, and wherein the one        or more detected droplets are introduced into the layer of fluid        on the first surface of the substrate.    -   80. The method of 76 or 77, wherein the carrier fluid is an        aqueous fluid and the droplets include a fluid which is        immiscible with the carrier fluid.    -   81. The method of 80, wherein the substrate includes on a first        surface a layer of aqueous fluid which is miscible with the        carrier fluid and immiscible with the fluid included by the        droplets, and wherein the one or more detected droplets are        introduced into the layer of aqueous fluid on the first surface        of the substrate.    -   82. The method of any one of 76-81, wherein the sorter includes        a flow channel including a gapped divider including a separating        wall which extends less than the complete height of the flow        channel.    -   83. A method of affixing a droplet to a substrate, the method        including:        -   delivering a droplet in a first carrier fluid from a            microfluidic device, through an orifice, to a substrate            surface;        -   positioning the droplet in a second carrier fluid on the            substrate surface; and        -   affixing the droplet to the substrate surface via a force.    -   84. The method of 83, wherein the first and second carrier fluid        are the same.    -   85. The method of 83, wherein the force is selected from a        gravitational force, an electrical force, a magnetic force, and        combinations thereof.    -   86. The method of 85, wherein the force is a magnetic force.    -   87. The method of 85, wherein the force is an electrical force.    -   88. The method of 87, wherein the electrical force is a        dielectrophoretic force.    -   89. The method of 89, wherein the substrate includes a plurality        of channels filled with a conductive liquid or solid material        and an insulating sheet positioned between the plurality of        channels and the carrier fluid, wherein the plurality of        channels are patterned to generate an electric field gradient        above the insulating sheet upon application of a voltage, and        wherein the method includes applying a voltage to one or more of        the plurality of channels sufficient to generate the electrical        field gradient, wherein the electrical field gradient produces a        dielectrophoretic force sufficient to affix the droplet to the        substrate surface.    -   90. The method of 89, wherein the substrate includes a plurality        of channels filled with a conductive liquid or solid material        and an insulating sheet positioned between the plurality of        channels and the carrier fluid, wherein the plurality of        channels are patterned to generate a dielectrophoretic force        sufficient to affix the droplet to the substrate surface.    -   91. The method of 89, wherein the substrate and the droplet have        net charges which are opposite in polarity.    -   92. The method of 83, wherein the wettability of the substrate        is sufficient to affix the droplet to the substrate via wetting        forces.    -   93. The method of 83, including modifying the wettability of the        substrate so as to be sufficient to affix the droplet to the        substrate via wetting forces.    -   94. The method of 89, wherein the substrate includes a plurality        of channels filled with a conductive liquid or solid material        and an insulating sheet positioned between the plurality of        channels and the carrier fluid, wherein the plurality of        channels are patterned to provide a plurality of electrode        features, and wherein the plurality of electrode features are        positioned relative to each other so as to provide positions on        the substrate surface capable of reducing droplet interfacial        energy when a voltage is applied to one or more of the plurality        of channels, and wherein the positions are sufficient to affix        the droplet to the substrate surface.    -   95. The method of 94, wherein at least one electrode feature is        positioned relative to at least one other electrode feature such        that there is a gap between the features, wherein the distance        of the gap is within an order of magnitude of the diameter of        the droplet.    -   96. The method of 95, including affixing the droplet in        proximity to the gap.    -   97. The method of 83, wherein the method includes applying        exogenous electromagnetic radiation sufficient to affix the        droplet to a specific location on the substrate surface.    -   98. A method of moving an affixed droplet on a substrate, the        method including:        -   delivering a droplet in a first carrier fluid from a            microfluidic device, through an orifice, to a substrate            surface;        -   positioning the droplet in a second carrier fluid on the            substrate surface; affixing the droplet to the substrate            surface via a force; and        -   modulating the force so as to move the droplet from its            affixed location to another location and/or applying a            second force, which is sufficient, either alone or in            combination with the modulated force, to move the droplet            from its affixed location to another location.    -   99. The method of 98, wherein the first and second carrier fluid        are the same.    -   100. The method of 98 or 99, wherein the substrate includes a        plurality of channels filled with a conductive liquid or solid        material and an insulating sheet positioned between the        plurality of channels and the carrier fluid, wherein the        plurality of channels are patterned to generate an electric        field gradient above the insulating sheet upon application of a        voltage, and wherein the method includes applying a voltage to        one or more of the plurality of channels sufficient to generate        the electrical field gradient, wherein the electrical field        gradient produced a dielectrophoretic force sufficient to affix        the droplet to the substrate surface.    -   101. The method of 100, including modulating the electrical        field so as to move the droplet from its affixed location to        another location.    -   102. The method of 98, wherein the method includes applying        exogenous electromagnetic radiation sufficient to move the        droplet from its affixed location to another location.    -   103. The method of 98, wherein the method includes introducing a        cross flow of fluid which is sufficient to move the droplet from        its affixed location to another location.    -   104. The method of 98, wherein the buoyancy of the droplet in        the second carrier fluid is modulated to move the droplet from        its affixed location to another location.    -   105. A method of adding reagents to a droplet, the method        including:        -   delivering a first droplet in a first carrier fluid from a            microfluidic device, through an orifice, to a substrate            surface;        -   positioning the droplet in a second carrier fluid on the            substrate surface;        -   affixing the droplet to the substrate surface via a force;        -   delivering a second droplet to the same location as the            first droplet affixed to the substrate surface or a location            adjacent the first droplet on the substrate surface; and        -   coalescing the first droplet and the second droplet such            that the contents of the first droplet and the second            droplet are combined.    -   106. The method of 105, wherein the first and second carrier        fluid are the same.    -   107. The method of 105 or 106, wherein multiple droplets are        delivered to the same location as the first droplet affixed to        the substrate surface or a location adjacent the first droplet        on the substrate surface, and wherein the multiple droplets are        coalesced with the first droplet such that the contents of the        first droplet and the multiple droplets are combined.    -   108. The method of any one of 105-107, wherein coalescence is        triggered via application of a force to one or more of the        droplets.    -   109. The method of any one of 105-107, wherein coalescence        occurs spontaneously.    -   110. The method of 108, wherein the force is an electrical        force.    -   111. A method of adding reagents to a droplet, the method        including:        -   delivering a droplet in a first carrier fluid from a            microfluidic device, through a first orifice, to a substrate            surface;        -   positioning the droplet in a second carrier fluid on the            substrate surface;        -   affixing the droplet to the substrate surface via a force;        -   inserting a second orifice fluidically connected to a            reagent source into the droplet; and        -   injecting via the second orifice one or more reagents into            the droplet.    -   112. The method of 111, wherein the first and second carrier        fluid are the same.    -   113. A method of affixing a droplet to a substrate, the method        including:        -   delivering a droplet in a first carrier fluid from a            microfluidic device, through an orifice, to a substrate            surface;        -   positioning the droplet in a second carrier fluid on the            substrate surface;        -   affixing the droplet to the substrate surface via a force;            and        -   recovering all or a portion of the affixed droplet.    -   114. The method of 113, wherein the first and second carrier        fluid are the same.    -   115. The method of 113 or 114, wherein the recovering includes        modulating one or more forces acting on the affixed droplet.    -   116. The method of 113 or 114, wherein the recovering includes        contacting the affixed droplet with a microfluidic orifice        fluidically connected to a suction device to recover all or a        portion of the affixed droplet from the substrate surface.    -   117. The method of 113 or 114, wherein the recovering includes        bringing in proximity to the affixed droplet a microfluidic        orifice fluidically connected to a suction device to recover the        affixed droplet from the substrate surface.    -   118. The method of 113 or 114, wherein the recovering includes        inserting into the affixed droplet a microfluidic orifice        fluidically connected to a suction device to recover all or a        portion of the contents of the affixed droplet.    -   119. The method of 113 or 114, wherein the recovering includes        shearing the affixed droplet from the substrate surface.    -   120. The method of 113 or 114, wherein the recovering includes        increasing the buoyancy of the affixed droplet such that        buoyancy forces acting on the affixed droplet are sufficient to        overcome the force affixing the droplet to the substrate        surface, thereby releasing the affixed droplet from the        substrate surface.    -   121. The method of 120, wherein increasing the buoyancy of the        affixed droplet includes increasing the volume of the affixed        droplet.    -   122. The method of 121, wherein the volume of the affixed        droplet is increased by injecting an aqueous fluid into the        affixed droplet.    -   123. The method of 113 or 114, wherein the recovering includes        modulating the force affixing the droplet to the substrate        surface such that the droplet is released from the substrate        surface.    -   124. The method of 123, wherein the modulating includes removing        the force.    -   125. The method of 113 or 114, wherein the droplet includes one        or more beads.    -   126. The method of 125, wherein the one or more beads include a        binding agent which selectively binds one or more materials        present in the droplet.    -   127. The method of 125 or 126, wherein the one or more beads are        buoyant within the droplet.    -   128. The method of 125 or 126 wherein the one or more beads are        selected from magnetic beads and conductive beads.    -   129. The method of any one of 125-128, including positioning        and/or concentrating the one or more beads in a first region of        the droplet leaving a second region which is relatively devoid        of beads.    -   130. The method of 129, including selectively recovering from        the droplet the one or more beads from the first region.    -   131. The method of 129, including selectively recovering        material from the second region of the droplet.    -   132. The method of any one of 113-131, including delivering the        recovered droplet or the recovered portion of the droplet to one        or more isolated containers via a delivery orifice.    -   133. A method of manipulating an affixed droplet, the method        including:        -   delivering a droplet in a first carrier fluid from a            microfluidic device, through an orifice, to a substrate            surface;        -   positioning the droplet in a second carrier fluid on the            substrate surface;        -   affixing the droplet to the substrate surface via a force;            and        -   modulating the immediate environment of the droplet, thereby            modulating the contents of the droplet.    -   134. The method of 133, wherein the first and second carrier        fluid are the same.    -   135. The method of 133 or 134, wherein the modulating includes        modulating a parameter selected from a chemical composition of        the immediate environment, a temperature of the immediate        environment, a pH of the immediate environment, a pressure of        the immediate environment, and a radiation level of the        immediate environment.    -   136. The method of any one of 133-135, wherein the substrate        surface is selectively permeable, the substrate includes a fluid        volume positioned beneath and in contact with the selectively        permeable substrate surface, and the immediate environment of        the droplet is modulated by modulating one or more of a chemical        composition of the fluid volume, a temperature of the fluid        volume, a pH of the fluid volume, a pressure of the fluid        volume, and a radiation level of the fluid volume.    -   137. The method of 136, wherein the fluid volume is a fluid        flow.    -   138. The method of any one of 133-137, wherein the substrate        includes patterned electrodes positioned beneath the fluid        volume.    -   139. The method of any one of 133-138, including storing the        affixed droplet under controlled environmental conditions for a        storage period, wherein the force is maintained during the        storage period.    -   140. The method of 139, wherein the controlled environmental        conditions include a constant temperature and/or pressure.    -   141. The method of any one of 133-140, including at least        partially solidifying the affixed droplet.    -   142. The method of 141, including removing the second carrier        fluid from the substrate surface.    -   143. The method of 142, wherein the second carrier fluid is        immiscible with the contents of the affixed droplet prior to the        at least partial solidification of the affixed droplet, and        wherein the method includes replacing the removed second carrier        fluid with a miscible fluid.    -   144. The method of 143, including modulating a chemical        composition of the miscible fluid, thereby modulating the        affixed droplet.    -   145. A method of manipulating an affixed droplet, the method        including:        -   delivering a droplet in a first carrier fluid from a            microfluidic device, through an orifice, to a substrate            surface;        -   positioning the droplet in a second carrier fluid on the            substrate surface;        -   affixing the droplet to the substrate surface via a force;        -   at least partially solidifying the affixed droplet;        -   removing the second carrier fluid from the substrate            surface, wherein the second carrier fluid is immiscible with            the contents of the affixed droplet prior to the at least            partial solidification of the affixed droplet;        -   replacing the removed second carrier fluid with a miscible            fluid; and modulating a chemical composition of the miscible            fluid, thereby modulating the affixed droplet.    -   146. The method of 145, wherein the first and second carrier        fluid are the same.    -   147. A method of porating a cell within an affixed droplet, the        method including:        -   delivering a droplet in a first carrier fluid from a            microfluidic device, through an orifice, to a substrate            surface, wherein the droplet includes a cell;        -   positioning the droplet in a second carrier fluid on the            substrate surface;        -   affixing the droplet to the substrate surface via a force;            and        -   porating the cell within the droplet.    -   148. The method of 147, wherein the first and second carrier        fluid are the same.    -   149. The method of 147 or 148, wherein the cell is porated using        electrical, chemical, or sonic means.    -   150. The method of any one of 147-148, including introducing one        or more nucleic acids into the porated cell.    -   151. The method of any one of 147-150, wherein the poration        occurs within the microfluidic device.    -   152. The method of any one of 147-150, wherein the poration        occurs after delivery through the orifice and prior to affixing        the droplet to the substrate surface.    -   153. The method of any one of 147-150, wherein the poration        occurs after the droplet is affixed to the substrate surface.    -   154. The method of any one of 147-149, wherein the poration        occurs in the microfluidic device prior to delivery through the        orifice.    -   155. The method of 154, wherein the droplet is delivered to the        substrate surface in proximity to a second droplet positioned on        the substrate surface, wherein the second droplet includes a        nucleic acid, and wherein the method includes merging the        droplet with the second droplet to contact the nucleic acid with        the porated cell.    -   156. A method of analyzing a droplet on a substrate, the method        including:        -   delivering a droplet in a first carrier fluid from a            microfluidic device, through an orifice, to a substrate            surface;        -   positioning the droplet in a second carrier fluid on the            substrate surface;        -   affixing the droplet to the substrate surface via a force;            and        -   detecting one or more components of the affixed droplet.    -   157. The method of 156, wherein the first and second carrier        fluid are the same.    -   158. The method of 156 or 157, wherein the detecting is        performed at a plurality of time points.    -   159. The method of 156 or 157, wherein the method includes        continuously detecting the one or more components of the affixed        droplet over a period of time.    -   160. The method of 158 or 159, wherein the method includes        detecting a change in the one or more components of the affixed        droplet.    -   161. The method of any one of 156-160, wherein the detecting        includes optically detecting the one or more components of the        affixed droplet.    -   162. The method of 161, wherein the optically detecting includes        detecting an absorbance of the one or more components of the        affixed droplet.    -   163. The method of 161, wherein the optically detecting includes        detecting a fluorescence of the one or more components of the        affixed droplet.    -   164. The method of any one of 156-160, wherein the detecting        includes detecting the one or more components of the affixed        droplet via one or more spectroscopic techniques.    -   165. The method of 164, wherein the one or more spectroscopic        techniques are selected from nuclear magnetic resonance (NMR)        spectroscopy, UV spectroscopy, and mass spectrometry.    -   166. The method of any one of 156-160, wherein the method        includes adding a suitable matrix-assisted laser        desorption/ionization (MALDI) matrix material to the droplet        either before or after fixation, and detecting one or more        components of the affixed droplet via MALDI.    -   167. The method of any one of 156-165, including recovering and        analyzing all or a portion of the affixed droplet.    -   168. A method of delivering discrete entities to a substrate,        the method including:        -   flowing a plurality of first discrete entities through a            first microfluidic device in a first carrier fluid, wherein            the first discrete entities are insoluble and/or immiscible            in the first carrier fluid, and wherein the first            microfluidic device includes a first delivery orifice;        -   directing the first carrier fluid and one or more of the            plurality of first discrete entities through the first            delivery orifice to the substrate;        -   flowing a plurality of second discrete entities through a            second microfluidic device in a second carrier fluid,            wherein the second discrete entities are insoluble and/or            immiscible in the second carrier fluid, and wherein the            second microfluidic device includes a second delivery            orifice;        -   directing the second carrier fluid and one or more of the            plurality of second discrete entities through the second            delivery orifice to the substrate; and        -   affixing the one or more of the plurality of first discrete            entities and the one or more of the plurality of second            discrete entities to the substrate.    -   169. The method of 168, wherein the first discrete entities        and/or the second discrete entities are droplets.    -   170. The method of 169, wherein the droplets include an aqueous        fluid, which is immiscible with the first carrier fluid and the        second carrier fluid.    -   171. The method of 170, wherein the substrate includes on a        first surface a layer of fluid which is miscible with the        carrier fluid and immiscible with the aqueous fluid, and wherein        the droplets are affixed to the first surface of the substrate        following introduction into the layer of fluid on the first        surface of the substrate.    -   172. The method of 169, wherein the first carrier fluid and the        second carrier fluid are aqueous fluids and the droplets include        a fluid which is immiscible with the first carrier fluid and the        second carrier fluid.    -   173. The method of 172, wherein the substrate includes on a        first surface a layer of aqueous fluid which is miscible with        the first carrier fluid and the second carrier fluid and        immiscible with the fluid included by the droplets, and wherein        the droplets are affixed to the first surface of the substrate        following introduction into the layer of aqueous fluid on the        first surface of the substrate.    -   174. The method of any one of 168-173, wherein the first carrier        fluid and the second carrier fluid are the same.    -   175. The method of any one of 168-174, wherein the first        microfluidic device and the second microfluidic device are        integrated with an automated system which selectively positions        the delivery orifices relative to the substrate, and wherein the        method includes selectively positioning via the automated system        the delivery orifices relative to the substrate to selectively        deliver the plurality of first discrete entities and the        plurality of second discrete entities to one or more locations        on or in proximity to the substrate.    -   176. The method of any one of 168-174, wherein the first        microfluidic device and the second microfluidic device are        integrated with an automated system which selectively positions        the substrate relative to the delivery orifices, and wherein the        method includes selectively positioning via the automated system        the substrate relative to the delivery orifices to selectively        deliver the plurality of first discrete entities and the        plurality of second discrete entities to one or more locations        on or in proximity to the substrate.    -   177. A method of delivering discrete entities to a substrate,        the method including:        -   flowing a plurality of discrete entities through a            microfluidic device in a carrier fluid, wherein the discrete            entities are insoluble and/or immiscible in the carrier            fluid, and wherein the microfluidic device includes a            plurality of delivery orifices;        -   directing the carrier fluid and a first one or more of the            plurality of discrete entities through a first delivery            orifice of the plurality of delivery orifices to the            substrate;        -   directing the carrier fluid and a second one or more of the            plurality of discrete entities through a second delivery            orifice of the plurality of delivery orifices to the            substrate; and        -   affixing the first one or more of the plurality of first            discrete entities and the second one or more of the            plurality of discrete entities to the substrate.    -   178. The method of 177, wherein the discrete entities are        droplets.    -   179. The method of 178, wherein the droplets include an aqueous        fluid, which is immiscible with the carrier fluid.    -   180. The method of 179, wherein the substrate includes on a        first surface a layer of fluid which is miscible with the        carrier fluid and immiscible with the aqueous fluid, and wherein        the droplets are affixed to the first surface of the substrate        following introduction into the layer of fluid on the first        surface of the substrate.    -   181. The method of 178, wherein the carrier fluid is an aqueous        fluid and the droplets include a fluid which is immiscible with        the carrier fluid.    -   182. The method of 181, wherein the substrate includes on a        first surface a layer of aqueous fluid which is miscible with        the carrier fluid and immiscible with the fluid included by the        droplets, and wherein the droplets are affixed to the first        surface of the substrate following introduction into the layer        of aqueous fluid on the first surface of the substrate.    -   183. The method of any one of 177-182, wherein the microfluidic        device is integrated with an automated system which selectively        positions the delivery orifices relative to the substrate, and        wherein the method includes selectively positioning via the        automated system the delivery orifices relative to the substrate        to selectively deliver the first one or more of the plurality of        discrete entities and the second one or more of the plurality of        discrete entities to one or more locations on or in proximity to        the substrate.    -   184. The method of any one of 177-182, wherein the microfluidic        device is integrated with an automated system which selectively        positions the substrate relative to the delivery orifices, and        wherein the method includes selectively positioning via the        automated system the substrate relative to the delivery orifices        to selectively deliver the first one or more of the plurality of        discrete entities and the second one or more of the plurality of        discrete entities to one or more locations on or in proximity to        the substrate.    -   185. A method of analyzing a droplet, the method including:        -   flowing a plurality of droplets through a microfluidic            device in a carrier fluid,        -   encapsulating or incorporating unique identifier molecules            into the plurality of droplets, such that each droplet of            the plurality of droplets includes a different unique            identifier molecule;        -   delivering the plurality of droplets in a first carrier            fluid from a microfluidic device, through an orifice, to a            substrate surface;        -   positioning the plurality of droplets in a second carrier            fluid on the substrate surface;        -   affixing the plurality of droplets to the substrate surface            via a force;        -   for each of the affixed plurality of droplets, recovering            all or a portion of the affixed droplet and the unique            identifier for each droplet;        -   analyzing the recovered droplets or recovered portions            thereof in conjunction with the unique identifier, wherein            results of the analysis are identified as specific to            material originating from particular droplets based on the            presence of the unique identifier.    -   186. The method of 185, wherein the first carrier fluid and the        second carrier fluid are the same.    -   187. The method of 185, wherein the unique identifier molecules        specifically bind to one or more materials present in the        plurality of droplets.    -   188. The method of 185, wherein no two droplets include the same        unique identifier molecule.    -   189. The method of 185, wherein the unique identifier molecules        include nucleic acids.    -   190. The method of 189, wherein the plurality of droplets        include nucleic acid molecules, one or more of the unique        identifier molecules are covalently bound to the nucleic acid        molecules, and wherein the method includes sequencing the        nucleic acid molecules together with the unique identifier        molecules, wherein the presence of the sequence of a unique        identifier molecule in the sequence read of a nucleic acid        molecule identifies the nucleic acid molecule as originating        from a particular droplet.    -   191. A method of performing quantitative PCR, the method        including:        -   partitioning a heterogeneous population of nucleic acids            into a plurality of droplets including an aqueous fluid;        -   encapsulating or incorporating quantitative PCR reagents            into the plurality of droplets;        -   flowing the plurality of droplets through a microfluidic            device in a carrier fluid, wherein the carrier fluid is            immiscible with the aqueous fluid;        -   directing the carrier fluid and a plurality of droplets            through a delivery orifice to a substrate;        -   affixing the plurality of droplets to the substrate, wherein            the substrate includes on a first surface a layer of fluid            which is miscible with the carrier fluid and immiscible with            the aqueous fluid, and wherein the plurality of droplets are            affixed to the first surface of the substrate following            introduction into the layer of fluid on the first surface of            the substrate;        -   incubating the affixed plurality of droplets under            conditions sufficient for amplification of nucleic acids;            and        -   detecting nucleic acid amplification over time.    -   192. The method of 191, wherein the encapsulating or        incorporating step occurs subsequent to the affixing step.    -   193. The method of 191 or 192, wherein the heterogeneous        population of nucleic acids is included by a plurality of cells,        and wherein the partitioning includes partitioning the plurality        of cells into the plurality of droplets including an aqueous        fluid.    -   194. The method of 193, wherein the method includes separate        cell lysis and nucleic acid amplification steps.    -   195. A method of sequencing single cell nucleic acids, the        method including:        -   partitioning a heterogeneous plurality of cells into a            plurality of droplets including an aqueous fluid, such that            each droplet includes not more than one cell;        -   subjecting the plurality of droplets to conditions            sufficient for lysis of the cells contained therein and            release of cellular nucleic acids;        -   encapsulating or incorporating unique nucleic acid            identifier molecules into the plurality of droplets, such            that each droplet of the plurality of droplets includes a            different unique nucleic acid identifier molecule;        -   linking the unique nucleic acid identifier molecules to one            or more cellular nucleic acids in the plurality of droplets            or to amplification products thereof;        -   flowing the plurality of droplets through a microfluidic            device in a first carrier fluid;        -   delivering the plurality of droplets in the first carrier            fluid from the microfluidic device, through an orifice, to a            substrate surface;        -   positioning the plurality of droplets in a second carrier            fluid on the substrate surface;        -   affixing the plurality of droplets to the substrate surface            via a force;        -   for each of the affixed plurality of droplets, recovering            all or a portion of the affixed droplet, including cellular            nucleic acids and the unique nucleic acid identifier for            each droplet;        -   sequencing nucleic acids from the recovered droplets or            recovered portions thereof together with the unique            identifier molecules, wherein the presence of the sequence            of a unique identifier molecule in the sequence read of a            nucleic acid molecule identifies the nucleic acid molecule            as originating from a particular cell.    -   196. The method of 195, wherein the subjecting step occurs        subsequent to the affixing step.    -   197. The method of 195, wherein the encapsulating or        incorporating step and the linking step occur subsequent to the        affixing step.    -   198. The method of any one of 195-197, wherein the method        includes separate cell lysis, nucleic acid amplification, and        linking steps.    -   199. A method of synthesizing a polymer on a substrate, the        method including:        -   flowing a first droplet including a first droplet fluid            through a microfluidic device in a carrier fluid, wherein            the first droplet includes a first polymer or a first            monomer;        -   directing the carrier fluid and the first droplet through a            delivery orifice to the substrate;        -   affixing the first droplet to the substrate wherein the            substrate includes on a first surface a layer of fluid which            is miscible with the carrier fluid and immiscible with the            first droplet fluid, and wherein the first droplet is            affixed to the first surface of the substrate at a            predetermined location following introduction into the layer            of fluid on the first surface of the substrate;        -   flowing a second droplet through the microfluidic device in            the carrier fluid, wherein the second droplet includes a            second polymer or a second monomer;        -   directing the carrier fluid and the second droplet through            the delivery orifice to the first droplet affixed at the            predetermined location;        -   incubating the first and second droplets under conditions            sufficient for the contents of the first and second droplets            to come into contact and for the first polymer or first            monomer to form a covalent bond with the second polymer or            monomer, thereby generating a synthesized polymer.    -   200. The method of 199, wherein the incubating includes        incubating the first and second droplets under conditions        sufficient for droplet coalescence.    -   201. The method of 199 or 200, wherein the synthesized polymer        is a polypeptide.    -   202. The method of 199 or 200, wherein the synthesized polymer        is a nucleic acid.    -   203. A method of analyzing a droplet on a substrate, the method        including:        -   partitioning a molecular library including a plurality of            library members into a plurality of droplets including an            aqueous fluid;        -   delivering the plurality of droplets in a first carrier            fluid from a microfluidic device, through an orifice, to a            substrate surface;        -   positioning the droplets in a second carrier fluid on the            substrate surface;        -   affixing the droplets to the substrate surface via a force;            and        -   performing one or more reactions in the affixed droplets            with the library members;        -   detecting the results of the one or more reactions in the            affixed droplets and/or recovering all or a portion of the            affixed droplets for further analysis.    -   204. The method of 203, wherein the first and second carrier        fluid are the same.    -   205. A method of printing microarrays, the method including:        -   delivering a plurality of droplets in a first carrier fluid            from a microfluidic device, through an orifice, to a            substrate surface, wherein each of the plurality of droplets            includes a molecule;        -   positioning the droplets in a second carrier fluid on the            substrate surface;        -   affixing the droplets at predetermined locations to the            substrate surface via a force;        -   incubating the substrate under conditions suitable for            chemical bonding of the molecules included by the affixed            droplets to the substrate surface, thereby providing an            array of substrate-bound molecules.    -   206. The method of 205, wherein the first and second carrier        fluid are the same.    -   207. A method of in situ sequencing, the method including:        -   flowing a plurality of droplets through a microfluidic            device in a carrier fluid,        -   encapsulating or incorporating unique nucleic acid            identifier molecules into the plurality of droplets, such            that each droplet of the plurality of droplets includes one            or more copies of a different unique nucleic acid identifier            molecule;        -   delivering the plurality of droplets in a first carrier            fluid from a microfluidic device, through an orifice, to a            surface of a tissue substrate;        -   positioning the plurality of droplets in a second carrier            fluid on the surface of the tissue substrate;        -   affixing the plurality of droplets to the surface of the            tissue substrate via a force;        -   incubating the tissue substrate under conditions sufficient            for the unique nucleic acid identifier molecules from each            affixed droplet to bind to nucleic acids contained within            the tissue substrate in proximity to the affixed droplet;        -   sequencing the unique nucleic acid identifier molecules and            the nucleic acids to which they are bound; and        -   identifying and/or quantitating, using the unique nucleic            acid identifier molecules, nucleic acids contained within            the tissue substrate at locations corresponding to locations            where particular droplets were affixed.    -   208. The method of 207, wherein the first and second carrier        fluid are the same.    -   209. A method of manipulating cells or embryos, the method        including:        -   flowing a plurality of droplets through a microfluidic            device in a carrier fluid, wherein each droplet of the            plurality of droplets includes an aqueous fluid and a            fertilized egg cell or embryo, and wherein the carrier fluid            is immiscible with the aqueous fluid;        -   directing the carrier fluid and the plurality of droplets            through a delivery orifice to a substrate;        -   affixing the plurality of droplets to the substrate, wherein            the substrate includes on a surface thereof a layer of fluid            which is miscible with the carrier fluid and immiscible with            the aqueous fluid, and wherein the plurality of droplets is            affixed to the surface of the substrate following            introduction into the layer of fluid on the surface of the            substrate;        -   detecting within the affixed plurality of droplets the            development of one or more embryos; and        -   selecting and recovering an embryo from the affixed            droplets.    -   210. A method of manipulating cells or embryos, the method        including:        -   flowing a plurality of droplets through a microfluidic            device in a carrier fluid, wherein each droplet of the            plurality of droplets includes an aqueous fluid and an            unfertilized egg cell, and wherein the carrier fluid is            immiscible with the aqueous fluid;        -   directing the carrier fluid and the plurality of droplets            through a delivery orifice to a substrate;        -   fertilizing one or more of the egg cells in the plurality of            droplets;        -   affixing the plurality of droplets to the substrate, wherein            the substrate includes on a surface thereof a layer of fluid            which is miscible with the carrier fluid and immiscible with            the aqueous fluid, and wherein the plurality of droplets are            affixed to the surface of the substrate following            introduction into the layer of fluid on the surface of the            substrate;        -   detecting within the affixed droplets the development of an            embryo; and        -   selecting and recovering specific embryos from the affixed            droplets.    -   211. The method of 210, wherein the fertilizing occurs after the        affixing.    -   212. A droplet printer including:        -   a microfluidic device including one or more droplet makers            and one or more flow channels, wherein the one or more flow            channels are fluidically connected to the one or more            droplet makers and configured to receive one or more            droplets therefrom;        -   a delivery orifice fluidically connected to one or more of            the one or more flow channels; and        -   an automated system integrated with the delivery orifice,            wherein the automated system (a) selectively positions the            delivery orifice in proximity to a substrate during            operation or (b) selectively positions the substrate in            proximity to the delivery orifice during operation, such            that a droplet can be ejected from the delivery orifice and            deposited on the substrate.    -   213. The droplet printer of 212, wherein the microfluidic device        includes a droplet sorter which selectively sorts droplets in        one or more of the one or more flow channels for delivery        through the delivery orifice.    -   214. The droplet printer of 212 or 213, wherein the microfluidic        device includes or is integrated with a temperature control        module which is capable of modulating the temperature of a        carrier fluid in the one or more flow channels.    -   215. The droplet printer of 214, wherein the temperature control        module is a thermal cycler.    -   216. The droplet printer of any one of 212-215, including a        detection means capable of detecting one or more droplets or one        or more droplet components in one or more of the one or more        flow channels.    -   217. The droplet printer of 216, wherein the detection means is        an optical imager.    -   218. The droplet printer of any one of 212-217, wherein the        sorter includes a flow channel including a gapped divider        including a separating wall which extends less than the complete        height of the flow channel.    -   219. A system including:        -   a droplet printer as set forth in any one of 212-217;        -   a substrate surface for receiving one or more droplets            deposited by the delivery orifice of the droplet printer;            and        -   one or more of:            -   (a) a temperature control module operably connected to                the droplet printer,            -   (b) a detection means operably connected to the droplet                printer,            -   (c) an incubator operably connected to the droplet                printer, and            -   (d) a sequencer operably connected to the droplet                printer; and        -   a conveyor configured to convey the substrate from a first            droplet receiving position to one or more of (a)-(d).    -   220. A substrate including:        -   a substrate surface including an immiscible phase fluid; and        -   an ordered array of droplets positioned in the immiscible            phase fluid, wherein the droplets are affixed to the            substrate surface, and wherein the ordered array of droplets            includes at least 10,000 individual droplets.    -   221. The substrate of 220, wherein the ordered array of droplets        includes at least 50,000 individual droplets.    -   222. The substrate of 221 wherein the ordered array of droplets        includes at least 100,000 individual droplets.    -   223. The substrate of 221 wherein the ordered array of droplets        includes at least 1,000,000 individual droplets.    -   224. The substrate of any one of 220-223, wherein the substrate        has a length of 128 mm or less and a width of 85 mm or less.    -   225. An electrode array system including:        -   an array of individually controllable electrodes embedded in            a substrate material; a power source; and        -   a controller, wherein the controller is configured to            selectively enable or disable an electrical connection            between the power source and each individually controllable            electrode in the array thereby providing an active or            inactive electrode respectively, and wherein, each active            electrode is capable of affixing a discrete entity to a            surface of the substrate material in proximity to the active            electrode when said discrete entity is deposited in            proximity to the active electrode.    -   226. The method of any one of 1-39, wherein one or more of the        plurality of discrete entities includes a plurality of        materials, and wherein the method includes subjecting one or        more of the affixed discrete entities including the plurality of        materials to conditions sufficient for assembly of the plurality        of the materials.    -   227. The method of 226, wherein the plurality of materials        includes a plurality of microparticles and/or nanoparticles.    -   228. The method of 226, wherein the plurality of materials        includes one or more metals.    -   229. The method of 226, wherein the plurality of materials        includes one or more semiconductor materials.    -   230. The method of 226, wherein the plurality of materials        includes one or more organic materials.    -   231. The method of 226, wherein the plurality of materials        includes one or more nucleic acids.    -   232. The method of 226, wherein the plurality of materials        includes one or more hydrogel materials.    -   233. The method of 226, wherein the plurality of materials        includes one or more liquid materials.    -   234. The method of 226, wherein the plurality of materials        includes one or more materials having a shape selected from a        sphere, a rod, a polyhedron, or a star.    -   235. The method of 226, wherein the plurality of materials        includes one or more materials having a surface coating.    -   236. The method of 235, wherein the surface coating is selected        from a charged coating, a hydrophilic coating, a hydrophobic        coating, and a coating including one or more molecular        recognition elements.    -   237. The method of 226, wherein the plurality of materials        includes one or more monomers and/or polymers.    -   238. The method of any one of 226-237, wherein the assembly        includes one or more covalent bonding interactions between the        plurality of materials.    -   239. The method of any one of 226-237, wherein the assembly        includes one or more non-covalent bonding interactions between        the plurality of materials.    -   240. The method of any one of 226-239, wherein subjecting the        one or more of the affixed discrete entities including the        plurality of materials to conditions sufficient for assembly of        a plurality of the materials includes exposing the one or more        of the affixed discrete entities including the plurality of        materials to light, an increase or decrease in temperature, a        magnetic force, an electric field (e.g., a frequency modulated        electric field), a catalyst, an enzyme (e.g., an enzyme        catalyst), a depletion force, and/or conditions sufficient for        self-assembly of the plurality of materials.    -   241. The method of any one of 226-240, including screening the        assembled materials for one or more properties.    -   242. The method of 241, wherein the one or more properties are        selected from conductivity; interactions with electromagnetic        radiation, e.g., visible light, UV or IR, such as index of        refraction or light scattering; fluorescence; magnetic        properties; interactions, e.g., binding interactions, with        biological components or entities (e.g., cells (e.g., bacteria        or mammalian), fungi, or viruses); catalytic properties;        buoyancy; and density.    -   243. A microfluidic device including:        -   an inlet channel;        -   a first outlet channel in fluid communication with the inlet            channel;        -   a second outlet channel in fluid communication with the            inlet channel;        -   a dividing wall separating the first outlet channel from the            second outlet channel, wherein the dividing wall includes a            first proximal portion having a height which is less than            the height of the inlet channel and a second distal portion            having a height which is equal to or greater than the height            of the inlet channel.    -   244. The microfluidic device of 243, including an electrode        configured to selectively apply an electric field in the inlet        channel upstream of the dividing wall.    -   245. The microfluidic device of 243 or 244, wherein the height        of the first proximal portion is from about 10% to about 90% of        the height of the inlet channel.    -   246. The microfluidic device of any one of 243-245, wherein the        length of the first proximal portion is equal to or greater than        the diameter of a microdroplet to be sorted by the microfluidic        device.    -   247. The microfluidic device of 246, wherein the length of the        first proximal portion is from about 1× to about 100× of the        diameter of a microdroplet to be sorted by the microfluidic        device.    -   248. The microfluidic device of 247, wherein the length of the        first proximal portion is from about 20× to about 30× of the        diameter of a microdroplet to be sorted by the microfluidic        device.    -   249. The microfluidic device of any one of 243-248, including a        collection reservoir in fluid communication with the first        outlet channel and a waste reservoir in fluid communication with        the second outlet channel.    -   250. A system including a microfluidic device as set forth in        any one of 244-249, and an optical detector configured to detect        an optical property of one or microdroplets in the inlet channel        upstream of the location of the application of the electric        field by the electrode.    -   251. A method of sorting microdroplets, the method including:        -   flowing a plurality of microdroplets through an inlet            channel of a microfluidic device in a carrier fluid;        -   detecting via a detector a property of one or more of the            plurality of microdroplets in the inlet channel;        -   applying an electric field to the inlet channel to            selectively deflect one or more of the plurality of            microdroplets into a first outlet channel in fluid            communication with the inlet channel or a second outlet            channel in fluid communication with the inlet channel based            on the detection of the property, wherein the microfluidic            device includes a dividing wall separating the first outlet            channel from the second outlet channel, wherein the dividing            wall includes a first proximal portion having a height which            is less than the height of the inlet channel and a second            distal portion having a height which is equal to or greater            than the height of the inlet channel.    -   252. The method of 251, wherein the property is an optical        property.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); nt,nucleotide(s); and the like.

Example 1 Fabrication and Testing of Microfluidic Nozzle and PatternedElectrode Substrate

A microdroplet printing system was built and tested using the schemedisplayed in FIG. 8. A droplet microfluidic print head, including acompact microfluidics droplet sorter modified with an exit nozzle, issuspended above the stage of an inverted microscope. Droplets flowingthrough the sorter are fluorescently labeled and detected within thedevice by a laser coupled to external detection optics. When a desireddroplet is detected, it is actively sorted to the nozzle and directed toa target surface. A constant background flow of carrier fluid (oil)brings the droplet in close contact with the dielectrophoretic trap. Acustomized substrate with biopolar electrodes patterned into its surfaceis placed on the xy stage of the microscope and serves as a target forthe deposition of droplets. Specific regions on the substrate with highelectric field gradients serve as dieletrophoretic traps for droplets bycausing movement of droplets towards, and wetting onto these regions. Inthis implementation, the nozzle of the print head is held stationary,while the substrate is translated horizontally.

FIG. 9 shows preliminary results from a droplet printing example. Theprint head and the network of dielectrophoretic traps are visible and inthe plane of the image. A series of droplets printed to the surface arevisible along the top of the image.

Construction of the Microfluidic Print Head: The microfluidic print headcontains a modified version of a dielectrophoresis-actuated sorter. Thesorter used is this example is designed to sort droplets withapproximately 80 μm diameters. The sorter geometry is molded in PDMSusing soft lithography techniques known in the art. The device geometryis trimmed down to a 2 cm×2 cm square, punched for fluidic access, andplasma bonded to a glass coverslip. The “sort” exit of the sorter iscoupled to a lateral exit from the device. A 250 μm OD/125 μm IDpolyethylene tube is inserted into this exit channel and glued in placeusing two part epoxy, creating a nozzle to direct droplets towards theprinting substrate. The top surface of the sorter is plasma bonded to a1″×3″ glass slide to enable anchoring of the sorter to a xyzmicromanipulator. This micromanipulator enables the placement of the tipof the print nozzle within 100 μm of the printing substrate surface.

Fabrication of the Printing Substrate: The printing substrate includestwo paired networks of electrodes that produce a grid ofdielectrophoretic traps on a planar surface as shown in FIG. 4. Theelectrodes are saltwater filled channels, electrified by an externalpower source, a scheme that has been shown to be effective indielectrophoretic-based microfluidics devices. The network of electrodesshown in FIG. 4 is molded in PDMS, using standard soft lithographytechniques. The device is punched for fluidic access and plasma bondedonto a glass slide with the molded geometric features facing up. Thechannels are then sealed from the top with a 25 μm thick piece of kaptontape. This sealing film should generally be relatively thin, since themagnitude of the electric field, and therefore the size of thedielectrophoretic force diminish with distance from the electrifiedfeatures. A rim of silicone caulking is deposited on the surface of thesealed device to maintain a thin layer of oil on top of thedielectrophoretic trap array. When the substrate is in use, the sealedchannels are filled with saltwater via pressurized syringes. An ACgenerator taken from a fluorescent lamp ballast is attached to thesyringe needles, and is used to apply about 1500 V at 30,000 Hz AC tothe saltwater channels.

Preliminary Experiments: Proof of concept experiments for themicrodroplet printer were performed to demonstrate the function of amicrofluidic sorter based print head in combination with adielectrophoretic trap substrate. An external dropmaker was used tocreate an emulsion composed of 80 μm aqueous droplets in oil. Theaqueous phase included PBS dyed with 40 μM fluorescein dye. The emulsionwas reinjected into the print head using a syringe pump. Specializedsoftware was developed to use the sorter as a drop on demand device,where a droplet with a desired set of fluorescent properties can besorted to the print nozzle when a button within the software interfaceis manually pressed. After a sorted drop was affixed to the printsubstrate, the xy stage of the inverted microscope was moved manually tothe next grid location. The droplets in FIG. 9 were printed using thistechnique, demonstrating the feasibility of this printing technology.

Example 2 Improved Sorting Architecture for High-Speed Sorting ofMicrodroplets

Described herein is a microfluidic design that permits 30 kHz dropletsorting with >99% accuracy. This tenfold rate increase compared to thefastest available droplet sorters enables ˜108 droplets to be sorted perhour and over a billion per day. Indeed, with the describedarchitecture, sorting speed is not limited by the physical mechanism ofsorting (even at Ca˜1) but rather by the electronics that detect thedroplets; with faster electronics, even faster sorting is anticipated.

The devices were fabricated using soft lithography ofpoly(dimethylsiloxane) (PDMS) moulded from device masters. The masterswere created from two sequential layers (11 μm and 19 μm thick) ofphotoresist (MicroChem, SU-8 3010) spun onto a silicon wafer. UncuredPDMS consisting of a 10:1 polymer to cross-linker mixture (Dow Corning,Sylgard 184) was poured onto the master, degassed, and baked at 85° C.for 2 hours. The PDMS mould was then cut and peeled from the master,punched with a 0.75 mm Harris Uni-core for inlet ports, and plasmabonded to a 1 mm thick, 10:1 PDMS slab to ensure a strong bond. Thebonded PDMS device was then baked at 85° C. for 10 min. The bottom ofthe all-PDMS device was then plasma bonded to a glass slide to providestructural support and rigidity. To enable immediate usage of the devicewith water-in-oil emulsions, a hydrophobic surface treatment wasperformed by flushing with Aquapel, clearing with pressurized air andbaking at 85° C. for an additional 30 min.

The primary innovation that allows for the increase in sorting speed byover an order of magnitude is the replacement of the impermeable wallthat usually divides the collection and waste channels with a gappeddivider. The gapped divider, which reaches only part way from thechannel ceiling to floor, allows droplets to squeeze into anenergetically unfavourable region (11 μm tall) between the sort channels(30 μm tall). Due to the droplet Laplace pressure, small lateraldisplacements above or below the sorter centre line grow as the dropletstravel downstream, pushing them fully into the nearest channel. Theprocess is shown in the schematic of FIG. 10, Panel (a), with a crosssection of the squeezed drop in the gapped divider inset. It is alsodepicted in the still in FIG. 10, Panel (b) taken from a high speedmovie of 25 μm droplets sorted at 22 kHz. This is ten-fold faster thanconventional sorters, which use hard wall dividers that split dropletsat similar flow rates due to shear at the divider edge (FIG. 10, Panel(c)). Splitting does not occur if droplets are displaced sufficientlybeyond the divider before they reach its starting edge; however, at highflow rates, the large electric fields utilized break the droplets apart(FIG. 10, Panel (d)). By contrast, when the gapped divider is used, thedroplets experience less shear and are able to gradually enter onechannel intact.

Another factor that may be important for achieving maximum sorting speedis the minimization of the oil spacer flow rate. Proper droplet spacingis important in allowing the droplets to be interrogated and sortedindividually, but too much oil increases capillary number and limitssorting speed. To minimize oil flow rate, a narrow, 50 μm-wide sortingjunction with a wide electrode was used, which exerted a constant forceon the droplets over a long distance. A second gapped divider was alsoimplemented at the entrance of the sorting junction (section at the leftof FIG. 10, Panels (a) and (b), which pinned incoming droplets againstthe upper wall so that the full channel was used during sorting and nooil was wasted. This divider operated by diverting high flow-ratecarrier oil, utilized to properly space droplets, from the reinjectionchannel into a lower channel carrying the bias oil used to tune lateraldrop position downstream. The combined oil flow from below pinnedincoming drops to the upper channel wall; without such a design, thedroplets move to the center of the channel.

The gapped dividers allow for the maximization of sorting speed, butother features may be important to ensure sorting accuracy. For example,as flow rates increase to sort faster, inlet pressures grow causing thedroplet filter to bow (FIG. 11, Panel (a)). Bowing widens the filtergaps permitting dust to pass that may clog the device. It also causesdroplets to pack in vertical layers, leading to irregular spacing (FIG.11, Panel (b)) and possible sorting errors. To address these issues, analternate design was used with the filter in the same shallow layer asthe gapped divider (drop inlet in FIG. 11, Panel (a)) rather than thetaller layer of the rest of the sorting junction (coloured grey). Thefilter still bowed under the pressure, but the gaps remained smallenough to remove debris. Moreover, as the droplets approached theinjection channel, they were forced into a monolayered, single-file linefor even spacing (FIG. 11, Panel (c)). Evidence of bowing can be seen inthe open areas of the filter, where deformation around the postsappeared as non-uniform shading and where droplets stacked vertically inmultiple layers (FIG. 11, Panel (d)).

After spacing, the droplets travelled to the sorter, as shown in FIG.11, Panel (a) and expanded in FIG. 10, Panels (a) and (b). There, thedroplets were scanned by a laser and their fluorescence measured. A saltwater electrode (2M NaCl) connected to a high voltage amplifier appliedthe electric field that sorted the droplets. The moat, a grounded saltwater electrode bordering the device, generated the field gradientnecessary for dielectrophoretic deflection and limited stray fields thatcould cause unintended droplet merger in the filter. Once sorted, thedivided populations travelled down two parallel channels with similarhydrodynamic resistance. Because the negatively sorted population wasoften much larger than the positively sorted, the negative channelexperienced greater flow resistance from droplet drag. To equilibratepressures and enable controlled dispensing into the collectionreservoirs, a shallow series of parallel channels was included near theoutlet (magnified in FIG. 11, Panel (e)) to allow oil, but not droplets,to move between outlets and equilibrate small pressure differentials.This also made the sorter less sensitive to small differences in theoutlet tube heights, which could generate a gravitational back-pressurethat could interfere with the droplet sorting.

To demonstrate the effectiveness of the fast sorter, it was used to sorta test emulsion including two droplet populations: a dim “negative”population and a bright “positive” population. The positive dropletsincluded phosphate-buffered saline with 1.4% by volume 0.5 μm latexbeads (Sigma Aldrich, L3280) to make them appear dark in the opticalmicroscopy images (FIG. 10, Panel (b)) and 0.75% fluorescent yellow 0.03μm latex beads (Sigma Aldrich, L5150) to make them brightly fluorescent.The negative droplets were phosphate-buffered saline with 0.13%fluorescent yellow beads, making them dimly fluorescent so that they toocould be detected by the drop detector. To create sufficient emulsionfor several hours of sorting at 30 kHz, the emulsions were generatedusing serial droplet splitting, formed initially as 50 μm droplets thatwere each halved three times to produce 8 droplets 25 μm in diameter.This enabled the generation of droplets at 2 mL/hr for the aqueousphase, approximately five times faster than could be achieved with aflow focusing generator.

To sort the emulsion, the droplets were injected into the device at 0.7mL/hr, with the drop spacing oil and drop position-tuning bias oil eachat 7 mL/hr. That corresponded to an average flow velocity of 3 m/sthrough the 30 μm×50 μm sorter cross section. The fluorinated oil (3M,HFE-7500) and 1% PEG-PFPE amphiphilic block copolymer surfactantcombined for a drop interfacial tension of 4 mN/m and a nearly matchedwater-oil dynamic viscosity of 0.1 mPa-s, giving a very large Ca of 0.8at that flow. The fluorescence was generated by a 473 nm laser (CNILasers), filtered at 517±10 nm by a bandpass filer (Semrock), andmeasured by a photomultiplier tube (PMT, Thorlabs, PMM02). The signalwas analysed by an FPGA (NI, PCI-7833R) with custom LabVIEW software.Droplets falling within the user-defined thresholds were sorted via anamplified pulse (Trek 609E-6) from the FPGA, transmitted into the devicevia a salt water electrode. To visualize the sorting and capture highspeed videos, the device was illuminated with infrared light that didnot overlap with the droplet fluorescence and imaged with a fast camera(Phantom, Miro M310) at a 50 kHz frame rate.

The fluorescence signals and pre- and post-sorted droplet populationsfrom an hour-long, ˜30 kHz sorting run (equivalent to processing over108 drops) are shown in FIG. 12. As the droplets passed through theexcitation laser, their emitted fluorescence was detected by the PMT,which outputted a voltage proportional to the intensity of the emittedlight. The semi-periodic drop fluorescences, as detected by the PMT, areshown in a time series in blue in FIG. 12, Panel (a), with thecorresponding sorting pulses in red. The PMT had a bandwidth of 0-20kHz, such that frequency components above this range were attenuated bya factor proportional to their fold-increase over 20 kHz. PMTs withhigher frequency response are commercially available and can beimplemented to detect droplets more quickly. The PMT voltages wererecorded at 200 kHz, a sampling period of 5 μs, which was approximatelythe time a droplet spends in the detector region. The individual dropletsignals, despite being broadened in time and attenuated in amplitude bythe limited PMT bandwidth, were nevertheless still well above the noisefloor and distinguishable as shown in the time trace.

Positive droplets were identified as those whose fluorescence was abovea PMT threshold of 0.15 V and whose temporal width at the threshold was<50 μs, which excluded large, merged droplets. When a positive dropletwas detected, the computer outputted a 1 V, 33 μs rectangular pulseamplified with edge rounding to 1 kV by the 13 kHz-bandwidth, highvoltage amplifier. The detection laser was positioned ahead of theelectrode so that, after identifying a droplet, an immediately-appliedpulse corresponded to the moment the droplet was directly opposite theelectrode, which was optimal for sorting. To estimate sorting speed, thedroplet rate in the time series of an hour long run was measured byidentifying peaks and, additionally, by measuring the maximum of theFourier transform, which was centred on 29±1 kHz (FIG. 12, Panel (a),inset). The sorting rates of conventional microfluidic devices areten-fold less than this and, for comparison, fall within the red band inthe left of the inset.

To confirm accurate droplet sorting, the pre- (FIG. 12, Panel (b)),positive- (FIG. 12, Panel (c)), and negative-sort (FIG. 12, Panel (d))droplet populations were imaged with fluorescence microscopy. Thepre-sort population was 6.4% bright, in agreement with the fraction ofpositives detected with the PMT time traces. The positive population was99.3% and the negative 0.2% bright. The false positives (dim droplets inthe positive population) were abnormally large (most were >3 times themean droplet volume, FIG. 12, Panel (c)) and were likely merged dropsthat were too large for the device design. The false negatives (brightdroplets in negative population) were abnormally small (<2 times themean droplet volume), which likely led to a proportionally smallerdielectrophoretic force and inadequate deflection during sorting. Inmost cases, sorting errors thus resulted from polydispersity in thestarting emulsion, suggesting that higher accuracy requires more uniformemulsions. This is difficult to achieve because most emulsions, nomatter the care taken to generate and handle them, will contain rareinstances of droplets that merged or split and are thus abnormally largeor small. Filtration of the emulsion prior to sorting may improve this,but requires additional steps that can result in even more merger andsplitting.

The described device achieved sorting rates that rival those offluorescence-activated cell sorters, which can sort at tens ofkilohertz. Recently, small microfluidic droplets (10-20 μm) have beensorted at 10-15 kHz using these FACS methods. However, this required adouble emulsification step in which the water-in-oil droplets weresuspended as water-in-oil-in-water double emulsions in an aqueouscarrier compatible with FACS. This may not be appropriate for allapplications since double emulsions are generally less stable thansingle emulsions and, in addition, tend to be more permeable to smallmolecules, which can leach out of the droplets over time. In instancesin which these issues are important, fast microfluidic droplet sortingis valuable.

As discussed herein, the present disclosure provides a microfluidicdevice that accurately sorts droplets at 30 kHz, ten times faster thanexisting droplet sorters. Pushing the rate higher is possible but mayrequire faster electronics. The speed of the described droplet sorterwill allow sorting of emulsions with unprecedented numbers of droplets.This will be valuable for applications in protein engineering and cellbiology, in which the target droplets or cells are extremely rare in thepopulation. Such enrichment is important, for example, for enhancingenzymes through droplet-based microfluidic directed evolution or forisolating very rare circulating tumor cells from blood cells.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this disclosure that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

Accordingly, the preceding merely illustrates the principles of theinvention. It will be appreciated that those skilled in the art will beable to devise various arrangements which, although not explicitlydescribed or shown herein, embody the principles of the invention andare included within its spirit and scope. Furthermore, all examples andconditional language recited herein are principally intended to aid thereader in understanding the principles of the invention being withoutlimitation to such specifically recited examples and conditions.Moreover, all statements herein reciting principles, aspects, andembodiments of the invention as well as specific examples thereof, areintended to encompass both structural and functional equivalentsthereof. Additionally, it is intended that such equivalents include bothcurrently known equivalents and equivalents developed in the future,i.e., any elements developed that perform the same function, regardlessof structure. The scope of the present invention, therefore, is notintended to be limited to the exemplary embodiments shown and describedherein. Rather, the scope and spirit of present invention is embodied bythe appended claims.

1. A method of delivering discrete entities to a substrate, the methodcomprising: flowing a plurality of discrete entities through amicrofluidic device in a carrier fluid, wherein the discrete entitiesare insoluble and/or immiscible in the carrier fluid, wherein themicrofluidic device comprises a sorter; sorting, via the sorter, the oneor more of the plurality of discrete entities to be delivered throughthe delivery orifice to the substrate from the plurality of discreteentities; directing the carrier fluid and the one or more of theplurality of discrete entities through the delivery orifice to thesubstrate; and affixing the one or more of the plurality of discreteentities to the substrate.
 2. The method of claim 1, wherein the one ormore of the plurality of discrete entities are affixed to the substratevia a force, wherein the force is selected from a gravitational force,an electrical force, a magnetic force, and combinations thereof.
 3. Themethod of claim 2, comprising storing the affixed entity undercontrolled environmental conditions for a storage period, wherein theforce is maintained during the storage period.
 4. The method of claim 3,wherein the controlled environmental conditions comprise a constanttemperature and/or pressure.
 5. The method of claim 2, wherein the forceis an electrical force.
 6. The method of claim 5, wherein the electricalforce is a dielectrophoretic force.
 7. The method of claim 1, whereinthe discrete entities are droplets.
 8. The method of claim 7, whereinthe droplets are affixed to the substrate via wetting.
 9. The method ofclaim 7, wherein the droplets comprise an aqueous fluid, which isimmiscible with the carrier fluid.
 10. The method of claim 9, whereinthe substrate comprises on a first surface a layer of fluid which ismiscible with the carrier fluid and immiscible with the aqueous fluid,and wherein the droplets are affixed to the first surface of thesubstrate following introduction into the layer of fluid on the firstsurface of the substrate.
 11. The method of claim 7, wherein the carrierfluid is an aqueous fluid and the droplets comprise a fluid which isimmiscible with the carrier fluid.
 12. The method of claim 11, whereinthe substrate comprises on a first surface a layer of aqueous fluidwhich is miscible with the carrier fluid and immiscible with the fluidcomprised by the droplets, and wherein the droplets are affixed to thefirst surface of the substrate following introduction into the layer ofaqueous fluid on the first surface of the substrate.
 13. The method ofclaim 1, wherein the discrete entities are affixed to the substrate viainterfacial tension.
 14. The method of claim 1, wherein the discreteentities have a dimension of from about 1 to 1000 μm.
 15. The method ofclaim 14, wherein the discrete entities have a diameter of from about 1to 1000 μm.
 16. The method of claim 1, wherein the discrete entitieshave a volume of from about 1 femtoliter to about 1000 nanoliters. 17.(canceled)
 18. The method of claim 1, wherein the sorter comprises aflow channel comprising a gapped divider comprising a separating wallwhich extends less than the complete height of the flow channel.
 19. Themethod of claim 1, wherein the plurality of discrete entities isoptically scanned prior to the sorting.
 20. The method of claim 19,wherein the sorter comprises an optical fiber configured to applyexcitation energy to one or more of the plurality of discrete entities.21. The method of claim 20, wherein the sorter comprises a secondoptical fiber configured to collect a signal produced by the applicationof excitation energy to one or more of the plurality of discreteentities.
 22. The method of claim 20, wherein the optical fiber isconfigured to apply excitation energy to one or more of the plurality ofdiscrete entities and collect a signal produced by the application ofthe excitation energy to one or more of the plurality of discreteentities.
 23. The method of claim 19, wherein the sorting is based onresults obtained from the optical scan.
 24. The method of claim 1,wherein the sorter is an active sorter.
 25. The method of claim 1,wherein the sorter is a passive sorter.
 26. The method of claim 24,wherein the sorting comprises sorting via dielectrophoresis.
 27. Themethod of claim 24, wherein the sorter comprises one or moremicrofluidic valves, and wherein the sorting comprises sorting viaactivation of the one or more microfluidic valves.
 28. The method ofclaim 1, wherein the discrete entities are droplets, the microfluidicdevice comprises a selectively activatable droplet maker which formsdroplets from a fluid stream, and wherein the method comprises formingone or more of the plurality of discrete entities via selectiveactivation of the droplet maker.
 29. The method of claim 1, wherein theplurality of discrete entities comprises discrete entities which differin composition.
 30. The method of claim 1, wherein the microfluidicdevice is integrated with an automated system which selectivelypositions the delivery orifice relative to the substrate, and whereinthe method comprises selectively positioning via the automated systemthe delivery orifice relative to the substrate to selectively deliverthe one or more of the plurality of discrete entities to one or morelocations on or in proximity to the substrate.
 31. The method of claim1, wherein the microfluidic device is integrated with an automatedsystem which selectively positions the substrate relative to thedelivery orifice, and wherein the method comprises selectivelypositioning via the automated system the substrate relative to thedelivery orifice to selectively deliver the one or more of the pluralityof discrete entities to one or more locations on or in proximity to thesubstrate.
 32. The method of claim 30, wherein the method comprisesdelivering a first member of the plurality of discrete entities to afirst location on or in proximity to the substrate and a second memberof the plurality of discrete entities to a second location on or inproximity to the substrate.
 33. The method of claim 32, wherein thefirst and second locations are the same.
 34. The method of claim 1,wherein one or more biological assays are performed in one or more ofthe discrete entities before and/or after delivery to the substrate. 35.The method of claim 1, wherein the temperature of one or more of thediscrete entities is controlled before and/or after delivery to thesubstrate.
 36. The method of claim 35, wherein one or more of thediscrete entities are thermalcycled before and/or after delivery to thesubstrate.
 37. The method of claim 1, wherein the members of theplurality of discrete entities which are not sorted for delivery throughthe delivery orifice to the substrate are recovered.
 38. The method ofclaim 37, wherein the recovered members of the plurality of discreteentities are recycled such that the method of claim 1 is repeated withthe recovered members of the plurality of discreet entities.
 39. Themethod of claim 38, wherein recovered members of the plurality ofdiscrete entities are continuously recycled during performance of themethod.
 40. The method of claim 1, wherein one or more of the pluralityof discrete entities comprises a cell.
 41. The method claim 40, whereineach member of the plurality of discrete entities comprises not morethan one cell.
 42. The method of claim 1, wherein one or more of theplurality of discrete entities comprises a nucleic acid.
 43. The methodof claim 1, wherein the method comprises encapsulating or incorporatingone or more reagents into the plurality of discrete entities.
 44. Themethod of claim 43, wherein the one or more reagents compriseamplification reagents.
 45. The method of claim 44, wherein theamplification reagents comprise Polymerase Chain Reaction (PCR)reagents. 46.-55. (canceled)